- Email: [email protected]
Functions of the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation
Accepted Manuscript Title: Functions of the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation Author: S. Matheis A. Yemelin D. Scheps K. Andresen S. Jacob E. Thines A.J. Foster PII: DOI: Reference:
S0944-5013(16)30747-9 http://dx.doi.org/doi:10.1016/j.micres.2016.12.010 MICRES 25980
To appear in: Received date: Revised date: Accepted date:
6-10-2016 23-12-2016 27-12-2016
Please cite this article as: Matheis S, Yemelin A, Scheps D, Andresen K, Jacob S, Thines E, Foster A.J.Functions of the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation.Microbiological Research http://dx.doi.org/10.1016/j.micres.2016.12.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Functions of the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation
Matheis, S.a‡, Yemelin, A.a*‡, Scheps, D.a, Andresen, K.b, Jacob, S.a, Thines, E.a,b and Foster A.J.a* a
Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (IBWF), Erwin-Schrödinger-Str. 56, D-67663 Kaiserslautern. b Institut für Mikrobiologie und Weinforschung, Johann-Joachim-Becherweg 15, D-55128 Mainz.
‡
both authors equally contributed to this work
*For correspondence. E-mail: [email protected], [email protected]
Keywords: Magnaporthe, conidiation, transcription factor, metabolic reprogramming, fluffy gene, melanization
Abstract The Magnaporthe oryzae genes FLB3 and FLB4, orthologues of the Aspergillus nidulans regulators of conidiation FlbC and FlbD, were inactivated. These genes encode C2H2 zinc finger and Myb-like transcription factors, respectively, in A. nidulans. Analysis of the resultant mutants demonstrated that FLB4 is essential for spore formation and that strains lacking this gene are fluffy in their colony morphology due to an inability to complete conidiophore formation. Meanwhile, FLB3 is required for normal levels of aerial mycelium formation. We identified genes dependent on both transcription factors using microarray analysis. This analysis revealed that the transcription of several genes encoding proteins implicated in sporulation in Magnaporthe oryzae and other filamentous fungi are affected by FLB3 or FLB4 inactivation. Furthermore, the microarray analysis indicates that Flb3p may effectively reprogramme the cell metabolically by repressing transcription of genes encoding biosynthetic enzymes and inducing transcription of genes encoding catabolic enzymes. Additionally, qRT-PCR was employed and showed that FLB3 and FLB4 transcripts are enriched in synchronously sporulating cultures, as were the transcripts of other genes that are necessary for normal conidiation, consistent with a role for their gene products in this process.
1
1. Introduction Sporulation (conidiation) is a critical phase in the disease cycle of the rice blast fungus Magnaporthe oryzae. The asexual spores (conidia) formed are the primary means by which rice blast disease is spread. Despite the importance of conidiation for this fungus, relatively little is understood regarding the genetic determinants of the process in comparison to the detailed knowledge of the prepenetrative growth phase. Conidiation in M. oryzae occurs when the fungus emerges from lesions, either through stomata or through tears in the cuticle (Howard and Valent, 1996). Conidia are formed from conidiogenous cells by a blastic mode of development (Cole, 1986; Howard, 1994). The conidia are arranged sympodially on the mature conidiophore, which normally bears three or more spores (Howard, 1994). Identification of the genetic determinants of conidiation has benefited from the availability of the Magnaporthe oryzae genome sequence and from large-scale mutagenesis programmes (Dean et al., 2005; Jeon et al., 2007). Two research groups have independently identified the MoHOX2/HTF1-homeobox transcription factor-encoding gene as essential for spore formation (Kim et al., 2009; Liu et al., 2010). Furthermore, the phosphotransferase-encoding gene MoYPD1 was recently also found to be somehow involved in conidiation. Loss of function mutants of MoYPD1 could not produce spores in various conditions (Jacob et al., 2015). These are the only genes identified to date whose mutation leads to a complete loss of conidiation in Magnaporthe; however, several mutants have been identified which show aberrant conidiophore or spore morphology and/or altered conidia production. The gene ACR1 was identified in a random mutagenesis project. ACR1 deletion leads to an abnormal conidiophore morphology: rather than the normal sympodial arrangement, the conidia of ∆acr1 mutants are produced in a chain and the gene was, thus, named ACR1 in view of this acropetal arrangement (Lau and Hamer, 1998; Nishimura et al., 2000). Further mutations affecting conidiation (con mutants) were identified by chemical or insertional mutagenesis (Shi and Leung, 1995; Shi et al., 1998). The ∆con5 and ∆con6 mutants are aconidial, the ∆con1 and ∆con2 strains display much reduced conidiation and produce misshapen spores, and the ∆con4 and ∆con7 mutants have abnormal spore morphology. In most cases, the CON gene affected has not yet been isolated or characterised, the exception being CON7, which was subsequently shown to encode a transcription factor which regulates the transcription of genes whose products influence the biogenesis of the M. oryzae cell wall (Odenbach et al., 2007). Further transcriptional regulators involved in sporulation include the MADS-box transcription factor MIG1. Mutants without MIG1 show reduced aerial mycelium growth and conidiation (Mehrabi et al., 2008). Recent large-scale mutagenesis programmes have identified many other sporulation mutants, however, a relationship between phenotype and implied genotype based on the recovery of DNA surrounding the insertion site has not been proved in most cases. Other genes whose products play an important role in the pre-penetrative growth phase have also been reported to be required for normal sporulation: notably the ∆mosnf1, ∆mps1 and ∆tps1 mutants all show an extreme reduction in sporulation (Foster et al., 2003; Xu et al., 1998; Yi et al., 2008). In the case of MoSNF1 and MPS1, which encode protein kinases required for normal virulence, these observations would suggest that signal transduction pathways controlling conidiation might share these components with some of the signalling pathways which regulate appressorium formation and/or function. The M. oryzae regulator of Gprotein signalling Rgs1p has recently been shown to negatively regulate conidiation via the G-subunit MagBp (Liu et al., 2007). Evidence for direct MagBp-Rgs1p interaction suggests that Rgs1p acts to accelerate the hydrolysis of GTP bound to MagBp and, therefore, regulates MagBp-dependent signalling negatively, hence leading to extreme reduction in conidiation of the ∆magB mutant and 2
the hyper-conidiation of the ∆rgs1 strain (Liu et al., 2007). The same study also revealed that a constitutively active form of the G-subunit MagAp subunit leads to reduced formation of aerial hyphae. The ultimate outputs for these signalling pathways in terms of changes in transcription are unclear. A recent study used microarray technology to identify genes up- and down-regulated during conidiation (Kim and Lee, 2012) and will serve as a useful resource for understanding conidiation in Magnaporthe. In comparison to the situation in the model ascomycetes Neurospora crassa and A. nidulans, relatively little is known about the genetic determinants of the conidiation process in M. oryzae. APSES transcriptional regulators, such as StuA in A. nidulans, are involved in conidiophore development, and loss of function mutants of MSTU1 in M. oryzae were found to produce a greatly decreased number of conidia (Nishimura et al., 2009). In the current study, we explored the consequences of the inactivation of the M. oryzae genes FLB3 and FLB4, which are predicted, based on sequence similarity, to encode the orthologues of the A. nidulans regulators of sporulation FlbCp and FlbDp. The implication of FlbDp in the conidiation-related process in A. nidulans has been elucidated previously, since the FlbD-deficient mutants were found to be abolished in conidiation. The FlbD gene was shown to be sufficient to direct conidiophore formation (Garzia et al., 2010; Wieser and Adams, 1995). Furthermore, the inactivation of FlbD revealed no alterations in the mycelial growth of A. nidulans, leading to the assumption that FlbD operates in a conidiation-specific manner. The FlbC gene, in its turn, was shown previously to apparently play a role in the transition from vegetative growth to development, regulating the time-specific expression of the conidiationrelated genes BrlA, AbaA and VosA. Thus, deletion of FlbC gene in A. nidulans resulted in a delayed conidiophore formation, a reduced expression of BrlA, AbaA and VosA, and a significantly reduced conidial germination, whereas the overexpression of the FlbC gene, on the contrary, activates the expression of conidiation-related genes and inhibits the hyphal growth (Kwon et al., 2010). We examined the role of FlbC and FlbD orthologues in M. oryzae to address the question whether A. nidulans and M. oryzae share common genetic regulators of molecular mechanism controlling asexual sporulation. The transcriptional profiling of FLB3- and FLB4-deficient mutants by microarray experiment should provide insights into the complexity of the regulation network of the conidiationrelated process by surveying the Flb3p- and Flb4p-dependent regulons. Our results indicate that M. oryzae Flb4p is essential for conidiation, while Flb3p is required for normal levels of aerial mycelium formation and, therefore, also for normal levels of sporulation. Comparison of the transcriptome of ∆flb3, ∆flb4 and wild type (WT) using microarray analysis revealed that the Flb3p and Flb4p regulons overlap significantly, but that Flb3p has a broader influence and may control a general response to nutrient limitation. The formation of aerial mycelium, together with an induction of the machinery for utilisation of five alternative carbon sources, is coupled to a reduction in the transcription of genes whose products would be expected to have biosynthetic activity.
2. Materials and Methods 2.1. Strains, materials, preparation of fungal cultures, phenotypic analysis The strains created in the present study were derived from M. oryzae WT strain 70-15 (Chao and Ellingboe, 1991), Guy11 or P1.2 (∆KU80) (Villalba et al., 2008). Fungal strains were maintained on complete medium (CM) agar plates (Talbot et al., 1993), which support good levels of sporulation. 3
We additionally used oatmeal agar (Crawford et al., 1986) and minimal media (MM) (Leung et al., 1995) supplemented with 1 % (w/v) of glucose, maltose, sucrose, fructose, glycerol, amino acids, gelatin, olive oil, cellulose, starch or xylan as a sole carbon source, respectively, to test for sporulation of mutants on different growth substrates. Standard methods for manipulation of DNA were used (Sambrook et al., 1989). Escherichia coli strain XL1-Blue (Stratagene) or TOP10 (Invitrogen) was used for bacterial transformations and for the cloning and maintenance of the constructs used. All reagents and equipment were sourced from Sigma unless otherwise stated. The growth of Magnaporthe strains for assays of sporulation, disease-related morphogenesis or extraction of RNA for microarray-based transcription analysis was performed according to methods described previously (Odenbach et al., 2007). Pathogenicity tests on roots of Oryza sativa were conducted consonant with Dufresne and Osbourn (2001) and pathogenicity towards rice plants was carried out as described before (Jacob et al., 2014; Kim et al., 2009; Zhou et al., 2009). Complete medium agar cultures were scraped using a sterile scalpel for the extraction of RNA from aerial mycelium, and ungerminated spores were processed immediately following collection from CM agar cultures for the extraction of RNA, as described previously (Odenbach et al., 2007). All experiments described were repeated at least three times. At least two independently isolated mutants for each gene disruption were analysed for all phenotypic tests.
2.2. Sequence analysis and comparisons Sequence comparisons were performed using the BLASTp interrogation (Altschul and Lipman, 1990) against the National Center for Biotechnology Information non-redundant or UniProt databases. Sequence comparisons with the Magnaporthe oryzae genome were conducted via theBroad Institute of Harvard and MIT. Domain searches were performed using the programme InterPro (accessible at http://www.ebi.ac.uk/Tools/pfa/iprscan5/). Predictions of cellular localisation were made using PSORTII (http://psort.hgc.jp/form2.html; Nakai and Kanehisa, 1992). The nuclear localisation signal (NLS) prediction was performed by cNLS Mapper (http://nls-mapper.iab.keio.ac.jp) using the cut-off score for prediction of > 3.5. The alignments with orthologous protein sequences predicted from A. nidulans were conducted using ClustalW version 2.0 (Larkin et al., 2007) considering “BLOSUM” as a cost matrix with gap open cost and gap extend cost of 10 and 0.1, respectively. Manual curation, annotation and visualisation of the sequences predicted were performed using the Geneious software package (http://www.geneious.com).
2.3. Gene inactivation and complementation constructs The primers FLB3ko-F: 5’-CGACCCTTACTCCCCCTCATCAAC-3’ and FLB3ko-R: 5’TGCAAGGCTCGTAGGGGTAGAAAA-3’ were used to generate a 1932 bp PCR product, which was ligated into the vector pGEMTeasy to generate pGEM-FLB3 for the inactivation of FLB3 gene. An MscI and AfeI double restriction of pGEM-FLB3 generated a blunt-ended vector fragment, which was then ligated to the HpaI HPT cassette from pCB1003 (Carroll et al., 1994) to give pGEM-FLB3-HPT. The latter was then cleaved with ApaI and SpeI and the purified insert ligated to a pCAMBIA0380 ApaI+SpeI fragment to create the final Agrobacterium-compatible gene inactivation vector pCAMBFLB3-KO (9611 bp). The initial gene inactivation vector for FLB4 was created by PCR amplification, resulting in a 1768 bp product from 70-15 genomic DNA of the FLB4 gene with flanking regions using 4
the primers FLB4ko-F: 5’-GCGCTTCTGCATTAACTCAACTCA-3’ and FLB4ko-R: 5’CATGGCCAAACAATAAAGGAAAAA-3’. The product was cloned into pGEMTeasy to give pGEM-FLB4, which was then cleaved with EcoRV and AfeI; the vector fragment was purified and ligated to the HpaI HPT cassette from pCB1003 to give pGEM-FLB4-HPT. The latter was then restricted with ApaI+SpeI and the purified insert was ligated to ApaI+SpeI cleaved pCAMBIA0380 to give the completed gene inactivation vector pCAMB-FLB4-KO (9840 bp). The construct pCOMP-FLB3 was generated by amplification of the WT gene locus from strain 70-15 using FLB3-comp-F: 5’-GTTAAATGGACATATCTCGTGGCA-3’ and FLB3-comp-R: 5’TTGGTATACACCTAGGCATCGCT-3’ for complementation of the FLB3-disrupted mutant. The PCR product was ligated to pGEMTeasy and then the insert excised using NotI and cloned into the vector pCAMBIA-ILV (Kramer et al., 2009) cut with PspOMI, allowing the final vector to be used for the transformation of hygromycin-resistant mutants and selecting them for resistance against chlorimuron ethyl. The primers FLB4-comp-F: 5’-GTTCCTGGCCGCTTTTTAGTCTTGTG-3’ and FLB4comp-R 5’-GCGCGTCCCTTTCCTTTGTTCTC-3’ were used to amplify a blunt-ended (Phusion polymerase generated) 3545 bp product, which was restricted directly with ApaI+SpeI and ligated into pCAMBIABAR (Kramer et al., 2009) and transformed into the ∆flb4 mutant for complementation of the ∆flb4 mutant with its native promoter-gene-terminator. The plasmids pFLB3-GFP and pFLB4-GFP were constructed for generation of GFP fusion proteins. pFLB3-GFP was made by amplification of the FLB3 promoter and ORF using the primers FLB3-comp-F: 5’-GTTAAATGGACATATCTCGTGGCA-3’ and FLB3BspHI-R: 5’-TCATGAAGTCAGAGTGATGGTCCTCGGAG-3’. The PCR product was first ligated into pGEMTeasy and then the insert from the resultant vector excised with ApaI and BspHI and ligated to NcoI and ApaI cleaved pAJF-GFP (Odenbach et al., 2007). The insert from the resultant vector pFLB3GFP was excised using ApaI and SpeI and ligated into the vector pCAMBIA-HPT to create pCAMBFLB3-GFP for the transformation of the WT. The 1893 bp PCR product was generated using primers FLB4-GFP-F: 5’-GTTTCCTGCACCCCTTTTCAAGTC-3’ and FLB-GFP-R: 5’CATGATGGCAGCCTGGCGGGTGAC-3’ for GFP fusion to Flb4p. The PCR product was ligated to pGEMTeasy and then excised with ApaI and BspHI. These primers remove the stop codon and add a BspHI restriction site allowing in-frame ligation to GFP using the NcoI and ApaI site in the vector pAJF-GFP. The insert from the resultant vector pFLB4-GFP was excised using ApaI and SpeI and ligated into the vector pCAMB-ILV to create pCAMB-FLB4-GFP, which could be transformed into the ∆flb4 mutant or WT strains.
2.4. Fungal transformations The completed vectors described above were transformed into the stated WT strains using Agrobacterium tumefaciens-mediated transformation (Rho et al., 2001). Selection was performed as described previously (Odenbach et al., 2007), except for sulphonylurea-resistant transformants, which were selected on MM agar using 100 µg ml-1 chlorimuron ethyl (Sigma).
2.5. Microarray based transcription analysis Microarray-based transcription profiling experiments were performed using version III Magnaporthe oryzae oligo microarrays (Agilent Technologies). The starting material for this study was derived from 5
the RNA isolated from aerial mycelium of the synchronous solid cultures of the WT 70-15 and ∆flb3- / ∆flb4-mutants, respectively. A spore suspension with a final concentration of 1x105 spores/ml was pipetted and plated evenly on CM-agar plates covered with sterile cellophane to obtain the mycelium biomass in the case of ∆flb3-mutant. An amount of 50 ml CM in a 100-ml Erlenmeyer flask was inoculated and incubated at 28 °C and 120 U/min for 3 d for the generation of synchronous aerial mycelium of the aconidial ∆flb4-mutant. Subsequently, 1 ml of the resulting culture was plated onto a surface of CM coated with sterile cellophane. After 5 d of incubation, the RNA isolation and quality control were performed, as described previously (Odenbach et al., 2007). All procedures regarding the microarray analysis were carried out consonant with the manufacturer’s recommendations. Four biological replications were performed, each time with a reciprocal labelling experiment (“dye-swap”). Data normalization and statistical analyses were performed using Bioconductor R package Limma (Smyth, 2004). Raw signals were background corrected by subtracting the local spot background. We applied two normalization steps: firstly, within the array, using global loess normalization, and secondly, between the arrays, using the normalizing method “Aquantile”. Differentially expressed genes were obtained by fitting a linear model for each gene into the series of arrays and applying empirical Bayes. The p-values for genes differentially regulated were adjusted using the Benjamini–Hochberg procedure for false discovery rate control (Benjamini and Hochberg, 1995). The results were filtered by cut-off values for the signal intensity A > 28, the logarithmic fold change > 1 and the p-value < 0.05. We have deposited the raw data at GEO under SuperSeries accession number GSE38329. All details are compliant with MIAME.
2.6. RNA isolation, cDNA amplification and qRT-PCR analysis The RNA template used for the cDNA synthesis was extracted from 70-15 mycelium grown either from point inoculated cultures or from cultures which were created by spreading a spore suspension over the plate surface. The RNA isolation and qRT-PCR analysis were conducted as described previously (Odenbach et al., 2007). The qRT-PCR reactions were performed using three independent biological replicates. The Pfaffl analysis model was applied (Pfaffl, 2001), under consideration of EFA1 (Elongation factor 1-alpha encoding gene MGG_03641) as a reference gene, for the quantification of qRT-PCR transcription analysis.
3. Results 3.1. Identification of potential conidiation-related genes of Magnaporthe oryzae The sequences of the A. nidulans transcription factors FlbCp (UniProt: O74256_EMEND) (Wieser et al., 1994) and FlbDp (UniProt: Q00658_EMEND) (Wieser and Adams, 1995) were used to interrogate the M. oryzae genome sequence (Dean et al., 2005) for the presence of potential orthologues. A predicted protein with significant similarity to the query sequence was identified in each case. In the case of FlbCp, the similarity between these likely orthologues is confined to the C-terminal half of the proteins, where 121 out of the last 193 amino acids are identical (63 %). The similarity between A. nidulans FlbDp and its M. oryzae orthologue Flb4p is limited to the N-terminus, where the two proteins have 56 out of the last 118 of amino acids in common (~47 %). In the case of both these pairs of orthologous proteins, the similarity is largely confined to the region where the predicted DNA 6
binding domains are located. The Magnaporthe genes which encode these predicted orthologues of A. nidulans – FlbCp and FlbDp – were named FLB3 (Broad ID: MGG_04699) and FLB4 (Broad ID: MGG_06898), respectively. Sequence analyses were performed to compare the domain structure of the potential M. oryzae proteins identified with their corresponding orthologues in A. nidulans (Fig. S1). FLB3 is predicted to encode a protein with 392 amino acids which has two C2H2 zinc finger domains (PFAM ID: PF00096) at the C-terminal end of the protein (amino acids 324-346 and 352376). Two C-terminally located C2H2 zinc finger domains are also present in A. nidulans FlbCp. The predicted M. oryzae Flb3p protein has one potential NLS at position 369-379 (LRRHRKVHKGD) and one identical sequence is also present at a similar location in A. nidulans FlbCp. The FLB4 gene is predicted to encode a protein with 321 amino acids. Two closely spaced Myb-like potential DNAbinding domains (PFAM ID: PF00249) are located between amino acids 14-65 and 68-116. A very similar N-terminal arrangement of Myb-like domains is present in A. nidulans FlbDp as well as in the N. crassa orthologue RCA-1p. The residues 117-141 within M. oryzae Flb4p are predicted to form a coiled region. A potential bipartite NLS (RQNKLKMAEKKQMAQRKAEQDAKAAAKMASI) is present between amino acids 113 and 143.
3.2. FLB3 and FLB4 gene products localise to the nucleus, validating their predicted role of transcription factors In frame fusions of the FLB3 and FLB4 open reading frames (ORFs) to the GFP-encoding eGFP gene were used to demonstrate that the fusion proteins localise to the nucleus. The Flb4-GFP fusion protein showed, on average, a very faint fluorescence in spores and mycelium; however, it was found to be nuclear localised in both cell types, as was the Flb3p-GFP fusion protein (Fig. 1). The low abundance of the Flb4-GFP fusion protein may be due to the very low transcript abundance we have recorded for FLB4 within mycelium and spores (data not shown). Together, these analyses suggested that M. oryzae Flb3p and Flb4p might be transcription factors and orthologues of the A. nidulans regulators of sporulation FlbCp and FlbDp, respectively.
3.3. FLB3 and FLB4 encode products which regulate discrete steps in asexual spore formation and control melanin biosynthesis in M. oryzae We created ∆flb3 and ∆flb4 mutants using targeted gene disruption to investigate whether the M. oryzae proteins Flb3p and Flb4p have a role in the regulation of sporulation in a similar manner to FlbCp and FlbDp of A. nidulans investigated previously. Several transformants were recovered for both genes. Evaluation by southern blot analysis indicated that the gene had been successfully replaced with the hygromycin phosphotransferase-encoding gene (HPT) by homologous recombination in each case (Fig. S2). The conidiation of the ∆flb3 and ∆flb4 mutants thus generated was compared to that of the WT strain. This analysis revealed the ∆flb4 mutant to be aconidial on all media we tested, while the ∆flb3 mutant showed reduced sporulation on CM agar plates (28 ± 4 % of that of the WT). Conidiophores or even conidia-like structures was not detectable in ∆flb4 mutant. The amount of aerial mycelium formed by the ∆flb4 mutant was comparable to that of the WT; however, the culture is white and fluffy, as the aerial mycelium formed fails to differentiate (Fig. 2A). A drastic reduction in the production of aerial mycelium was observed with the ∆flb3 mutant, giving the culture a flat appearance (Fig. 2A). Fewer conidiophores are formed by the ∆flb3 mutant 7
compared to the WT strain, however, displaying a normal morphology (Fig. 2B). Pigmentation of the mutants cultivated on CM agar medium also differed: the ∆flb3 strains being darker than WT, while the ∆flb4 strains lacked pigment and were white or only showed pigmented sectors during the initial growth phase (Fig. 2C). It was not possible to assess the virulence of the ∆flb4 mutant by normal means, as infection in Magnaporthe is dependent on appressoria, which are formed after conidia have germinated. Nevertheless, we attempted to see whether we could infect rice leaves by placing mycelial blocks onto the leaf surface. It has been reported that appressoria can arise directly from the mycelial fragments in the case of the ∆cos1 mutant of M. oryzae and with certain WT strains (Kim et al., 2009; Zhou et al., 2009). By using this method, lesion formation from inoculations with mycelium could be found using the WT strain and ∆flb4, but was not observed using the ∆flb4 strain. We also didn’t find any appressoria-like structures at the hyphae tips of the mycelium used for the plant assays. Magnaporthe strains are also able to infect the roots of their hosts inoculated with mycelial fragments (Sesma and Osbourn, 2004). Therefore, we also tested whether the ∆flb4 strain was affected in its ability to infect roots. This analysis showed that the inoculations with the mutant gave rise to lesions like those of the WT (Fig. S3). Although the mechanisms by which roots and leaves are infected differ (Sesma and Osbourn, 2004), these results are consistent with the view that Flb4p may be a sporulation-specific regulatory factor. The ∆flb3 mutants, meanwhile, were unaffected in their virulence to susceptible rice cultivar CO-39 or to the barley cultivar “golden promise”, and lesions on leaves (data not shown) or roots (Fig. S3) were like those of the WT control. We reintroduced the FLB3 and FLB4 genes under the control of their native promoters and transcriptional termination regions into the respective mutants to prove that the defects we had observed were due to the inactivation of the target genes and not to some unrelated mutation. In the case of the ∆flb3 mutant, ∆flb3/FLB3 complemented strains were created by ectopic integration of the FLB3 native promoter-ORF-terminator. These strains were restored to WT levels in their production of conidia and aerial mycelium, indicating that the phenotypes we had observed in ∆flb3 strains were, in fact, due to FLB3 inactivation (data not shown). Similarly, we could restore spore formation and pigmentation to the ∆flb4 mutant by reintroduction of the FLB4 gene with its native promoter or with the Flb4-GFP fusion protein, although in both cases, WT levels of sporulation were not seen (data not shown). To prove that the defects we had seen were not strain specific, we also performed the FLB4 disruption experiment in two further WT strains, Guy11 and P1.2 (Villalba et al., 2008), and recovered multiple FLB4-disrupted strains which were verified by southern blotting. As expected, the corresponding mutants were aconidial, fluffy and displayed markedly reduced pigmentation in every case. Together, our experiments provide convincing evidence that the product of FLB4 is essential for sporulation and that the FLB3 gene product is required for production of normal levels of aerial mycelium, conidiophores and, therefore, also for normal levels of conidiation in M. oryzae.
3.4. Identification of potential Flb3p- and Flb4p-regulated genes using microarray-based transcriptome analysis Because FLB3 and FLB4 are predicted to encode transcription factors important for conidiation in M. oryzae, it was of interest to identify genes whose transcription is dependent on these proteins. Transcript abundance within total RNA isolated from mycelium scraped from the surface of CM agar cultures of the mutants and the WT (70-15) was compared using microarray experiments (summarised in Fig. 3 and shown in detail in Table S1). In the case of the ∆flb3 mutant, 474 genes 8
were found whose transcript abundance was significantly reduced. In order to extract the maximal meaning from the results, we then manually annotated these genes by assigning a function based on the presence of conserved functional domains and/or the best meaningful match to the predicted protein using BLASTp interrogation against the UniProt database (Jain et al., 2009). We found this manual method of classifying the genes identified consonant with function to be superior to our attempts at automated annotation using GO terms. Our results revealed that there were several conidiation-related genes among the genes whose transcript abundance was decreased by FLB3 inactivation, suggesting their transcriptional regulation by an FLB3-encoded transcription factor. This gene set includes, for instance, MPG1 (MGG_10315), which encodes a hydrophobin associated with aerial mycelium and known to be required for normal sporulation and virulence in M. oryzae (Talbot et al., 1993; Talbot et al., 1996). The set also includes the ACR1 (MGG_09847) gene, already discussed in the introduction, orthologues of the N. crassa CON6 and CON8 (MGG_02246; MGG_00513) genes, which are described as conidiation-specific in this species (Berlin and Yanofsky, 1985; Roberts and Yanofsky, 1989), and the MoFLP1 (MGG_02884) gene, whose inactivation leads to a mutant phenotype similar to that observed in the case of the FLB3-disruption mutant (Liu et al., 2009). Another gene whose transcript showed reduced abundance consistently in the ∆flb3 mutant is predicted to encode the orthologue of the A. nidulans PhiA protein (MGG_00081), which is essential for phialide development in this species (Melin et al., 2003). Additionally, many genes had predicted products which were involved in the utilisation of diverse carbon sources (especially cellulose and fatty acids) or whose transporters of varied function showed reduced transcript abundance. Several of the predicted transcriptional regulators were also among the predicted products of genes whose transcript showed a decreased abundance in the ∆flb3 mutants; however, the transcription of the FLB4 gene was not affected by FLB3-inactivation. Interestingly, the gene TRA1 (MGG_10197), previously shown to encode a transcription factor required for normal adhesion, germination, appressorium formation and virulence, was also among the genes with reduced transcript abundance (Breth et al., 2013). At the same time, the microarray analysis revealed 315 genes with reproducibly increased transcriptional level in aerial mycelium of the FLB3-disruption mutant. Interestingly, factors with biosynthetic (including those involved in synthesis of fatty acids, amino acids and melanin) rather than catabolic activity dominated among the predicted products encoded by genes with increased transcript abundance in the ∆flb3 mutant. In the case of the ∆flb4 mutant, 107 genes were found to be significantly down-regulated and 40 genes displayed a reproducibly increased abundance. More than half of the genes significantly downregulated also displayed decreased transcript abundance in the ∆flb3 mutant, including the MoFLP1 and MPG1 genes mentioned above and the CHS7 gene encoding class VII chitin synthase, previously shown to be involved in appressorium development and virulence in M. oryzae (Odenbach et al., 2009). Notable exceptions were the melanin biosynthetic genes BUF1 (MGG_02252) and ALB1 (MGG_07219) (Chumley and Valent, 1990; Oh et al., 2008) which show decreased transcript abundance in the ∆flb4 mutant and increased abundance in the ∆flb3 strain, providing, at the same time, a likely explanation for the increased pigmentation of ∆flb3 and the decreased pigmentation of ∆flb4 strains. The predicted products of the genes RGS7 (MGG_11693) and RGS8 (MGG_13926) harbouring the “regulator of G protein signalling” domain (PS50132) exhibited a dramatic reduction in transcript abundance in the ∆flb4 mutant, whereas in the case of the ∆flb3 mutant, only RGS8 revealed a decreased amount of the corresponding transcripts. However, mutants lacking these factors were previously shown to be unaffected in asexual spore formation (Zhang et al., 2011). 9
3.5. Role of the FLB3 gene product during growth on different carbon sources As we had observed that a large set of genes encoding carbohydrate-active enzymes showed reduced transcript abundance in the ∆flb3 strain, we tested the growth and sporulation of this mutant on different carbon sources including glucose, fructose, maltose, gelatin, amino acids and olive oil (Fig. 4). The outcome of these tests was the observation that although sporulation of the mutant was reduced on most carbon sources tested, the level of sporulation was carbon sourcedependent, as more spores were produced compared to the WT when cultivating on MM maltose. Notably, a well conserved maltose transporter is the product of one gene whose transcript abundance was found to be increased in ∆flb3, providing a possible explanation for the increased sporulation of this strain on MM maltose. The results from these growth tests also imply that media that support low growth of the fungus are also not good substrates for spore production, such as olive oil. Olive oil leads to sparse growth of the fungal culture and to very low spore production, i.e. on MM with olive oil (data not shown) or on CM supplemented with olive oil and without glucose. This could presumably indicate that a certain level of biomass must be accumulated before sporulation is induced and/or that nutrient availability locally is sensed to set the sporulation process in motion and that this critical level is reached much later for a sparsely growing Magnaporthe colony. Conceivable triggers for sporulation within the lesions of the rice leaf might be multiple factors, for example, nutrient status, light (Lee et al., 2006), desiccation or oxidative stress (among others). Except for light, we have found no evidence that any of these acts induce sporulation in axenic cultures (data not shown).
3.6. Analysis of transcription of FLB3, FLB4 and genes identified as being dependent on their products during conidiation We looked at the transcription of the genes in the WT strain 70-15 studied during growth on CM agar plates under two different conditions: point-inoculated cultures, where the oldest mycelium is towards the centre of the plate, and what we have termed “synchronously conidiating cultures”, which were created by spreading a spore suspension over the whole of the plate. Synchronously conidiating cultures produced at least twofold more conidia after 9 d (Fig. 5A). Therefore, it is logical to conclude that the proportion of the colony engaged in sporulation is probably higher in synchronously conidiating cultures than in point-inoculated cultures. We believe that a comparison of transcript abundance between these two different conditions would be a useful means to assess whether a specific gene was induced during sporulation, as such genes would be expected to show a higher transcript abundance in synchronously conidiating cultures than in point-inoculated cultures. Indeed, we found that FLB3, FLB4, ACR1, MPG1, MoFLP1 and others showed higher transcript abundance in synchronously conidiating cultures (Fig. 5B). Meanwhile the transcript of a gene UVI1 (MGG_02647) that we have shown previously to be dispensable for spore formation (data not shown) displays no differential transcript abundance. Based on our results, we propose that synchronously conidiating cultures versus point-inoculated cultures might be a simple and effective means to search for sporulation-related factors by transcription profiling.
10
3.7. Comparison between microarray analysis investigating conidiation A study was published recently which examined transcriptional changes associated with sporulation from the KJ201 strain (Kim and Lee, 2012). The study clearly demonstrates that the transcription of both FLB3 and FLB4 is induced during sporulation, as we have proposed in the previous section. The study also compared microarray results between the WT and a HOX2-deleted strain which forms increased numbers of conidiophores, but no spores, and revealed that the transcription of both FLB3 and FLB4 is Hox2p-dependent. There are, therefore, several good reasons to compare the results of the current study with this published work. The transcription of those genes up-regulated during sporulation and, at the same time, showing decreased transcript abundance in our mutants might be considered especially good candidates for genes whose products are specifically involved in the sporulation process. Meanwhile, those genes whose transcripts show only decreased abundance in the ∆flb3, ∆flb4 or ∆hox2 mutants might represent those whose products have other roles during development (non-sporulation related activities of the transcription factors predicted). Additionally, the results of the work published to identify genes involved in sporulation (as the authors state) will include some transcriptional changes unrelated to sporulation, but related to the difference in age between the cultures used. Furthermore, genes with a low level of expression in more than one mutant (e.g. ∆hox2 and ∆flb4, whose phenotypes are most similar) and normally induced are good candidates for conserved sporulation factors. We performed a comparison of differentially expressed genes using Venn diagrams to identify sets of genes shared across the conditions mentioned previously and, thus, to deduce potential candidate genes for association with the conidiation process. General comparisons across the arrays are summarised in Table 1, and some of the pathogenicity- and conidiation-relevant genes known from previous studies and being shared across compared microarray results are visualized using Venn diagrams (Fig. S4A, B). The differentially regulated genes in common between different experiments are listed in Table S1, which also provides a list of potential candidate genes for further investigation, based on their hypothesized involvement in the conidiation process. We also performed a manual annotation of the results of Kim and Lee (2012), similar to that we made for our results (Table S2), to provide an objective comparison. In the case of the ∆flb4 mutant, a significant overlap between the FLB3 and FLB4 regulons was apparent, as one would anticipate based on the phenotypes of the respective mutants (~52 % of genes with reduced transcript abundance in the ∆flb4 mutant also exhibited reduced transcript abundance in the ∆flb3 mutant). Genes displaying a reduced transcript abundance in ∆flb3, but not in ∆flb4 will, therefore, be included, however, these will not be limited to those whose products are specifically involved in the initiation of aerial mycelium formation and not in later stages of the sporulation process. Our analysis of the FLB3- and FLB4-disruption stains had suggested a close link between the regulation of melanin biosynthesis and melanisation; however, a comparison with published microarrays makes it clear that this is not the case, and we propose that regulation of melanin biosynthesis may be a role different to and independent of the role of the Flb3p and Flb4p transcription factors in the sporulation process.
4. Discussion Relatively sparse information exists to date on the molecular basis of conidiation in M. oryzae, even though this process plays a critical role in the infection cycle. Thus, investigation and deepening our understanding regarding this process could provide a very important means to control the disease in 11
the field. Decoding of the signaling pathways involved in conidiation represents an effective approach to identify the genetic determinants of conidiation-associated processes, encompassing both the initiation and late phases of the conidiation. At the same time, much more extensive and detailed information exists on molecular mechanisms regarding the conidiation process in A. nidulans and N. crassa. The objectives of this study were to characterize fluffy gene homologues involved in conidiogenesis and their mechanism of action in M. oryzae. We characterized two fluffy gene homologues in M. oryzae and found that FlbDp and FlbCp are conserved regulators for conidiogenesis in A. nidulans and M. oryzae. Thus, disruption of the FlbD coding sequence in M. oryzae resulted in mutants lacking the conidiophore differentiation and the formation of spores, whereas the inactivation of the FlbC gene led to mutants unable to produce the intact conidiophores. Using the basic bioinformatic analysis, we were, furthermore, able to identify the other putative homologous genes in M. oryzae as known and described factors also involved in the conidiation process in A. nidulans and/or N. crassa, but with some exceptions. Hence, the observation that the gene encoding the transcription factor BrlAp, the unit of functional conidiation triad BrlAp-AbaApWetAp, playing a critical role in the activiation of sporulation pathway, is absent in the genome of M. oryzae is very interesting. Therefore, we suggest that, in the case of Magnaporthe, there must be other/additional mechanisms for the regulation of sporulation that might differ from the conserved regulation mechanisms. Although Kim et Al. have postulated in their study that the gene with the annotation number MGG_00501 encodes the BrlAp factor, whose expression would fit the Aspergillus nidulans hypothetical model very well, it is questionable whether it is indeed the gene, due to a weak similarity to its A. nidulans homologue (Liu et al., 2010). According to the A. nidulans model, FlbAp, FlbBp, FlbCp, FlbDp and FlbEp act as early regulators that activate the downstream operating central regulatory pathway in response to the product of FluG activity (Kim et al., 2009). The central genetic pathway of conidiation, in its turn, is composed of BrlAp, AbaAp and WetAp, coordinately regulating the order of gene activation during conidiophore development and spore maturation (Adams et al., 1998). Another example of the existence of the discrepancy concerning the conservation of regulation mechanisms of conidiation is provided by the VosA gene, previously functionally characterized in M. oryzae (Kim et al., 2014). The M. oryzae ΔvosA mutant has no defects in conidiation, although the A. nidulans VosA was previously shown to play a crucial role as a key regulator of the sporulation process (Ni and Yu, 2010). This observation provides the next evidence that gene pathways regulating conidiation may differ between fungal species and may derive from new mechanisms of gene regulation, rather than relying on changes concerning the biochemical function of the corresponding protein. Further investigation is necessary to define the genetic pathway and molecular mechanisms controlling conidiation in M. oryzae, and the understanding of the evolutionary-driven impact on the dynamics of developmental pathways is also essential for our understanding regarding the evolution of fungal adaptation concerning the pathogenicity-related processes. A microarray analysis was performed, based on the gene expression analysis of ∆flb3 and ∆flb4 mutants relative to that of the WT (70-15), to clarify the situation regarding the gene regulation of sporulation-associated processes in M. oryzae. Following this microarray analysis, we could reveal some useful first clues as to how the predicted products encoded by FLB3 and FLB4 may act to control sporulation. In line with their different roles in the process, some of the genes dependent on these factors were found to be in common, but also some of them were identified as unique to each transcription factor. The influence of Flb3p would generally seem to be broader. Flb3p obviously acts before Flb4p in the steps required to ultimately produce spores. Our results suggest that Flb4p function may be limited to the final steps in the 12
formation of conidiophores. Because aerial mycelium formation is reduced in the FLB3-disruption mutant, all subsequent steps in the sporulation process are also reduced and some degree of overlap in the microarray result was to be expected. Because the transcription of genes whose products influence carbohydrate metabolism was largely unaffected by the inactivation of FLB4, our results suggest that the influence of FLB3 on the transcription of such genes might be a function of Flb3p in addition to its role in aerial mycelium formation. In fact, it is logical to expect that the fungus would have some factors linking aerial mycelium formation (and ultimately spore formation) to nutrient status. The situation in Magnaporthe appears to be complex, as spore formation in axenic culture does not seem to be induced explicitly by starvation, similar to other fungi, and we believe that in its native environment, another factor (as yet unclear) triggers the sporulation of the fungus within lesions. Our results suggest that Flb3p may couple the initiation of aerial mycelium formation to the production of different carbohydrate-active enzymes: two logical responses to a limitation in preferred carbon sources. The results achieved in the current study allow us to draw up a hypothetical model of how the regulatory mechanism of conidiation could function in the case of M. oryzae (see Fig. 6).
Concluding remarks It would seem clear that Flb3p and Flb4p are responsible for transcriptional changes underlying the sporulation process. Flb3p appears to be implicated in the initiation of aerial mycelium formation and may also act to induce the production of factors required for the utilisation of alternative carbon sources. Flb4p may act later in sporulation and affects mainly conidiophore formation: no conidiophores or spores are formed in the absence of the protein. Melanisation may be repressed by the Flb3p function, whereas melanisation may be induced by the Flb4p function: mutants lacking FLB4 are almost completely non-pigmented. Some of the influences of these factors may operate indirectly, for example, through the control of transcription of other regulators, such as Acr1p. The roles of Flb3p and Flb4p proposed in our study are summarised in Fig. 6. The questions now are: what exactly is the timing of these transcriptional changes and what other factors are responsible for setting them in motion? In order to address these questions, we perhaps need to better understand the state of the fungal culture over time. At what point does aerial mycelium formation begin and is this simultaneous with the first appearance of conidiophores? What proportion of the aerial parts of the fungus on a solid medium are conidiophores and does this vary with time? Can we physically, genetically or pharmacologically uncouple aerial mycelium formation from sporulation (a prerequisite for differentiating between genes acting specifically on one process)? What genes are really controlled directly by the transcription factors studied to date? The current study shows the power, but also the limitations of microarray analysis. Transcriptional profiling over a time course could shed some light on the timing of events, but clearly, addressing the issues raised will require additional technologies/approaches (e.g. RNA-Seq). These are challenging questions, not least because, in contrast to the spore itself, the fungal culture is a complex and heterogeneous biomass. A final point worth some reflection is also how far can we extrapolate from studies of the fungal grown in axenic culture to the sporulation of the fungus where it really matters: in the field?
13
References Adams, T.H., Wieser, J.K., Yu, J.H., 1998. Asexual sporulation in Aspergillus nidulans. Microbiol. Mol. Biol. Rev. 62, 35–54. Altschul, S.F., Lipman, D.J., 1990. Protein database searches for multiple alignments. Proc. Natl. Acad. Sci. 87, 5509–5513. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Royal Stat. Soc. Ser. B. 57, 289–300. Berlin, V., Yanofsky, C., 1985. Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa. Mol. Cell Biol. 5, 849–855. Breth, B., Odenbach, D., Yemelin, A., Schlinck, N., Schröder, M., Bode, M., Antelo, L., Andresen, K., Thines, E., Foster, A.J., 2013. The role of the Tra1p transcription factor of Magnaporthe oryzae in spore adhesion and pathogenic development. Fungal Genet. Biol. 57, 11–22. Carroll, A., Sweigard, J., Valent, B., 1994. Improved vectors for selecting resistance to hygromycin. Fungal Genet. Newsl. 41, 22. Chao, C.C.T., Ellingboe, A.H., 1991. Selection for mating competence in Magnaporthe oryzae pathogenic to rice. Can. J. Bot. 69, 2130–2134. Chumley, F.G.,alent, B., 1990. Genetic analysis of melanin-deficient, nonpathogenic mutants of Magnaporthe grisea. Mol. Plant-Microbe Interact. 3, 135–143. Cole, G.T., 1986. Models of cell differentiation in conidial fungi. Microbiol. Rev. 50, 95–132. Crawford, M.S., Chumley, F.G., Weaver, C.G., Valent, B., 1986. Characterization of the heterokaryotic and vegetative diploid phases of Magnaporthe grisea. Genetics. 114, 1111–1129. Dean, R.A., Talbot, N.J., Ebbole, D.J., Farman, M.L., Mitchell, T.K., Orbach, M.J., Thon, M., Kulkarni, R., Xu, J.R., Pan, H., Read, N.D., Lee, Y.H., Carbone, I., Brown,D., Oh, Y.Y., Donofrio, N., Jeong, J.S., Soanes, D.M., Djonovic, S., Kolomiets, E., Rehmeyer, C., Li, W., Harding, M., Kim, S., Lebrun, M.H., Bohnert, H., Coughlan, S., Butler, J., Calvo, S., Ma, L.J., Nicol, R., Purcell, S., Nusbaum, C., Galagan, J.E., Birren, B.W., 2005. The genome sequence of the rice blast fungus Magnaporthe grisea. Nature, 434, 980–986. Dufresne, M., Osbourn, A.E., 2001. Definition of tissue-specific and general requirements for plant infection in a phytopathogenic fungus. Mol. Plant Microbe Interact. 14, 300–307. Foster, A.J., Jenkinson, J.M., Talbot, N.J., 2003. Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J. 22, 225–216, 235. Garzia, A., Etxebeste, O., Herrero‐García, E., Ugalde, U., Espeso, E.A. 2010. The concerted action of bZip and cMyb transcription factors FlbB and FlbD induces brlA expression and asexual development in Aspergillus nidulans. Mol. Microbiol. 75, 1314–1324. Howard, R.J., 1994. Cell biology of pathogenesis, in: Zeigler, S., Leong, S.A., Teng, P.S. (Eds.), Rice Blast Disease. CAB International, Wallingford, UK, pp. 3-22. Howard, R.J., Valent, B., 1996. Breaking and entering: Host penetration by the fungal rice blast pathogen Magnaporthe grisea. Annu. Rev. Microbiol. 50, 491–512.
14
Jacob, S., Foster, A.J., Yemelin, A., Thines, E., 2014. Histidine kinases mediate differentiation, stress response, and pathogenicity in Magnaporthe oryzae. Microbiologyopen 3, 668–87. Jacob, S., Foster, A.J., Yemelin, A., Thines, E., 2015. High osmolarity glycerol (HOG) signaling in Magnaporthe oryzae: Identification of MoYPD1 and its role in osmoregulation, fungicide action and pathogenicity. Fungal Biol. 119, 580–594. Jain, E., Bairoch, A., Duvaud, S., Phan, I., Redaschi, N., Suzek, B. E., Martin, M. J., McGarvey P., Gasteiger, E., 2009. Infrastructure for the life sciences: design and implementation of the UniProt website. BMC Bioinformatics 10, 1. Jeon, J., Park, S.Y., Chi, M.H., Choi, J., Park, J., Rho, H.S., Kim, S., Goh, J., Yoo, S., Choi, J., Park, J.Y., Yi, M., Yang, S., Kwon, M.J., Han, S.S., Kim, B.R., Khang, C.H., Park, B., Lim, S.E., Jung, K., Kong, S., Karunakaran, M., Oh, H.S., Kim, H., Kim, S., Park, J., Kang, S., Choi, W.B., Kang, S., Lee, Y.H., 2007. Genome-wide functional analysis of pathogenicity genes in the rice blast fungus. Nat Genet. 39, 561–565. Kim, H.J., Han, J.H., Kim, K.S., Lee, Y.H., 2014. Comparative functional analysis of the velvet gene family reveals unique roles in fungal development and pathogenicity in Magnaporthe oryzae. Fungal Genet. Biol. 66, 33–43. Kim, K.S., Lee, Y.H., 2012. Gene expression profiling during conidiation in the rice blast pathogen Magnaporthe oryzae. PLoS One. 7, e43202. Kim, S., Park, S.Y., Kim, K.S., Rho, H.S., Chi, M.H., Choi, J., Park, J., Kong, S., Park, J., Goh, J., Lee, Y.H., 2009. Homeobox transcription factors are required for conidiation and appressorium development in the rice blast fungus Magnaporthe oryzae. PLoS Genet. 5:e1000757. Kosugi, S., Hasebe, M., Tomita, M., Yanagawa, H., 2009. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc. Nat. Acad. Sci. 106, 10171– 10176. Kramer, B., Thines, E. Foster, A.J., 2009. MAP kinase signaling pathway components and targets conserved between the distantly related plant pathogenic fungi Mycosphaerella graminicola and Magnaporthe grisea. Fungal Genet. Biol. 46:667 – 681. Kwon, N.J., Garzia, A., Espeso, E.A., Ugalde, U., & Yu, J.H., 2010. FlbC is a putative nuclear C2H2 transcription factor regulating development in Aspergillus nidulans. Mol. Microbial. 77, 1203–1219. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson T.J., Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. Lau, G.W., Hamer, J.E., 1998. Acropetal: a genetic locus required for conidiophore architecture and pathogenicity in the rice blast fungus. Fungal Genet. Biol. 24, 228–239. Lee, K., Singh, P., Chung, W.C., Ash, J., Kim, T.S., Hang, L., Park, S., 2006. Light regulation of asexual development in the rice blast fungus, Magnaporthe oryzae. Fungal Genet Biol. 43, 694–706. Leung, H., Loomis, P., Pall, M.L., 1995. Transformation of Magnaporthe oryzae to phosphinothricin resistance using the bar gene from Streptomyces hygroscopicus. Fungal Genet. Newsl. 42, 41–43. Liu, H., Suresh, A., Willard, F.S., Siderovski, D.P., Lu, S., Naqvi, N.I., 2007. Rgs1 regulates multiple Gα subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism. EMBO J. 26, 690–700.
15
Liu, T.B., Chen, G.Q., Min, H., Lin, F.C., 2009. MoFLP1, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in Magnaporthe oryzae. JZhejiang Univ. Sci. B 10, 434–44. Liu, W., Xie, S., Zhao, X., Chen, X., Zheng, W., Lu, G., Xu, J-R., Wang, Z., 2010. A homeobox gene is essential for conidiogenesis of the rice blast fungus Magnaporthe oryzae. Mol. Plant Microbe Interact. 23, 366–375. Mehrabi, R., Ding, S., Xu, J.-R., 2008. MADS-box transcription factor mig1 is required for infectious growth in Magnaporthe grisea. Eukaryot. Cell 7, 791–799. Melin, P., Schnürer, J., Wagner, E.G., 2003. Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans. Fungal Genet. Biol. 40, 234–241. Nakai, K., Kanehisa, M., 1992. A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14, 897–911. Ni, M., Yu, J.H., 2007. A novel regulator couples sporogenesis and trehalose biogenesis in Aspergillus nidulans. PloS one, 2, e970. Nishimura, M., Fukada, J., Moriwaki, A., Fujikawa, T., Ohashi, M., Hibi, T., Hayashi, N., 2009. Mstu1, an APSES transcription factor, is required for appressorium-mediated infection in Magnaporthe grisea. Biosci. Biotechnol. Biochem. 73, 1779–1786. Nishimura, M., Hayashi, N., Jwa, N.S., Lau, G.W., Hamer, J.E., Hasebe, A., 2000. Insertion of the LINE retrotransposon MGL causes a conidiophore pattern mutation in Magnaporthe grisea. Mol. Plant-Microbe Interact. 8, 892–894. Odenbach, D., Breth, B., Thines, E., Weber, R.W.S., Anke, H., Foster, A.J., 2007. The transcription factor Con7p is a central regulator of infection-related morphogenesis in the rice blast fungus Magnaporthe grisea. Mol. Microbiol. 64, 293–307. Odenbach, D., Thines, E., Anke, H., Foster, A.J., 2009. The Magnaporthe grisea class VII chitin synthase is required for normal appressorial development and function. Mol. Plant Pathol. 10, 81–94. Oh, Y., Donofrio, N., Pan, H., Coughlan, S., Brown, D.E., Meng, S., Mitchell, T., Dean, R.A., 2008. Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae. Genome Biol. 9, R85. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45. Rho, H.S., Kang, S., Lee, Y.H., 2001. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol. Cells 12, 407–411. Roberts, A.N., Yanofsky, C., 1989. Genes expressed during conidiation in Neurospora crassa: characterization of con-8. Nucleic Acids Res. 17, 197–214. Sambrook J., Fritsch E.F., Maniatis T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sesma, A., Osbourn, A.E., 2004. The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi. Nature. 431, 582–586. Shi, Z., Christian, D., Leung, H., 1998. Interactions between spore morphogenetic mutations affect cell types, sporulation, and pathogenesis in Magnaporthe grisea. Mol. Plant-Microbe Interact. 11, 199–207.
16
Shi, Z., Leung, H., 1995. Genetic analysis of sporulation in Magnaporthe grisea by chemical and insertional mutagenesis. Mol. Plant-Microbe Interact. 8, 949–959. Smyth, G.K., 2004. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, 1–29. Talbot, N.J., Ebbole, D.J., Hamer, J.E., 1993. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5, 1575–1590. Talbot, N.J., Kershaw, M.J., Wakley, G.E., De Vries, O., Wessels, J., Hamer, J.E., 1996. MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. Plant Cell. 8, 985–999. Villalba, F., Collemare, J., Landraud, P., Lambou, K., Brozek, V., Cirer, B., Morin, D., Bruel, C., Beffa, R., Lebrun, M.H., 2008. Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for nonhomologous end joining. Fungal Genet. Biol. 45, 68–75. Wieser, J., Adams, T.H., 1995. flbD encodes a Myb-like DNA-binding protein that coordinates initiation of Aspergillus nidulans conidiophore development. Genes Dev. 9, 491–502. Wieser, J., Lee, B.N., Fondon, J. 3rd, Adams, T.H., 1994. Genetic requirements for initiating asexual development in Aspergillus nidulans. Curr. Genet. 27, 62–69. Xu, J.R., Staiger, C.J. Hamer, J.E., 1998. Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc. Natl. Acad. Sci. U. S. A. 95, 12713–12718. Yi, M., Park, J.H., Ahn, J.H., Lee, Y.H., 2008. MoSNF1 regulates sporulation and pathogenicity in the rice blast fungus Magnaporthe oryzae. Fungal Genet. Biol. 45, 1172–1181. Zhang, H., Tang, W., Liu, K., Huang, Q., Zhang, X., Yan, X., Chen, Y., Wang, J., Qi, Z., Wang, Z., Zheng, X., Wang, P., Zhang, Z., 2011. Eight RGS and RGS-like proteins orchestrate growth, differentiation, and pathogenicity of Magnaporthe oryzae. PLoS Pathog. 7, e1002450. Zhou, Z., Li, G., Lin, C. He, C., 2009. Conidiophore stalk-less1 encodes a putative zinc-finger protein involved in the early stage of conidiation and mycelial infection in Magnaporthe oryzae. Mol. Plant Microbe Interact. 22, 402–410.
17
Table 1: Comparison between microarray results of the current study and microarray results of Kim and Lee (2012)
during Conidiation*
in ∆flb4
in ∆hox2*
Up-regulated
Down-regulated
Up-regulated
Down-regulated
Up-regulated
Down-regulated
Down-regulated in ∆flb3 (474 genes)
18 % (85)
3.2 % (15)
0.2 % (1)
11.8 % (56)
5.7 % (27)
14 % (66)
Up-regulated in ∆flb3 (315 genes)
2.2 % (7)
15.9 % (50)
6.7 % (21)
3.2 % (10)
12.1 % (38)
2.5 % (8)
during Conidiation*
in ∆flb3
in ∆hox2*
Up-regulated
Down-regulated
Up-regulated
Down-regulated
Up-regulated
Down-regulated
Down-regulated in ∆flb4 (107 genes)
26 % (28)
3.7 % (4)
9.3 % (10)
52.3 % (56)
5.6 % (6)
15 % (16)
Up-regulated in ∆flb4 (40 genes)
0 % (0)
10 % (4)
52.5 % (21)
2.5 % (1)
17.5 % (7)
0 % (0)
Differentially expressed genes derived from the previous study by Kim and Lee (2012)
18
Figure Legends Fig. 1: Expression and cellular localisation of Flb3p::GFP and Flb4p::GFP in conidia and mycelium GFP-fusion to Flb3p and Flb4p indicates nuclear localisation of fusion proteins. Co-localisation with DAPIstained DNA was apparent for both Flb3p-GFP and Flb4-GFP proteins. Bar indicates 10 µm.
Fig. 2: Phenotypic features of the ∆flb3 and ∆flb4 mutants
A. Side view of cultures of WT (70-15) grown in CM and the ∆flb3 and ∆flb4 mutants shows that the ∆flb3 strain produces very little aerial mycelium/conidiophores, giving the culture a very flat profile. The ∆flb4 strain produces extensive tall growing aerial mycelium (without conidiophores), giving the culture a fluffy appearance. B. Conidiophores which are produced by the ∆flb3 strain resemble those of WT. Bar indicates 10 µm. C. Melanisation of the ∆flb3 strain is increased, while that of the ∆flb4 strain is decreased (most plates are totally white with rarely some pigmented regions during initial growth).
Fig. 3: Microarray results displayed by functional enrichment analysis Bar charts represent functional classification of products of the differentially regulated genes predicted to be Flb3p- and Flb4p-dependent. The products of the differentially expressed genes according to microarray analysis were manually annotated by assigning a function based on the presence of conserved functional domains and/or the best meaningful match to the predicted protein. Only the top nine enriched functionrelated terms are represented in the respective bar chart. A. Functional classification of the products of Flb3pdependent genes showing significant transcript abundance. Red bars represent significant functional categories for products of the down-regulated genes. Green bars indicate those for up-regulated genes.
B. Functional
classification of the products of Flb4p-dependent genes with significant transcript abundance. The colours of the corresponding bars are used in analogy to A. Full details of the microarray features, Broad IDs and their predicted gene products are given in Table S1.
Fig. 4: Conidiation of the FLB3-disruption mutant on varied carbon sources The number of spores per plate was assessed after incubation period of 11 d using six plates per condition; the experiment was repeated at least three times. Sporulation of the ∆flb3 mutant complemented by reintroduction of functional copy of the FLB3 gene is shown as a control, indicated as ∆flb3/FLB3. Sporulation of all strains on MM olive oil was too low to give reliable data.
Fig. 5: Sporulation of point-inoculated and synchronous cultures and use of these to analyse sporulation-related genes
A. Conidiation on two types of culture was tested: synchronous cultures which were inoculated using a spore suspension spread across the plate surface and point-inoculated cultures which were started by placing a mycelial block in the centre of the solid medium used (CM agar). Synchronous cultures produce more spores than point-inoculated cultures at later time points.
B. Quantitative reverse transcription polymerase chain
reaction (qRT-PCR) was used to assess the relative transcript abundance during sporulation using two different types of cultures of WT (70-15). Relative transcript abundance was assessed using the method of Pfaffl (2001) and EFA1 (MGG_03641) as a reference gene. Three biological replicates were performed. The transcripts of
19
FLB3 and FLB4 and the genes whose products are known mediators of sporulation-related process were significantly more abundant in the synchronous cultures.
Fig. 6: Model summarising the proposed functions for Flb3p and Flb4p Flb3p appears to be implicated in the initiation of aerial mycelium formation and may also act to induce the production of factors required for the utilisation of alternative carbon sources (both catabolic enzymes and transporters for uptake of their products). Melanisation may normally be repressed by Flb3p function. Flb4p may act later in sporulation and affects mainly conidiophore formation: no conidiophores or spores are formed in the absence of the protein. Melanisation may normally be induced by Flb4p function: mutants lacking FLB4 are almost completely non-pigmented. Some of the influences of these factors may operate indirectly, for example, through the control of transcription of other regulators, such as Acr1p.
20
S0944-5013(16)30747-9 http://dx.doi.org/doi:10.1016/j.micres.2016.12.010 MICRES 25980
To appear in: Received date: Revised date: Accepted date:
6-10-2016 23-12-2016 27-12-2016
Please cite this article as: Matheis S, Yemelin A, Scheps D, Andresen K, Jacob S, Thines E, Foster A.J.Functions of the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation.Microbiological Research http://dx.doi.org/10.1016/j.micres.2016.12.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Functions of the Magnaporthe oryzae Flb3p and Flb4p transcription factors in the regulation of conidiation
Matheis, S.a‡, Yemelin, A.a*‡, Scheps, D.a, Andresen, K.b, Jacob, S.a, Thines, E.a,b and Foster A.J.a* a
Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (IBWF), Erwin-Schrödinger-Str. 56, D-67663 Kaiserslautern. b Institut für Mikrobiologie und Weinforschung, Johann-Joachim-Becherweg 15, D-55128 Mainz.
‡
both authors equally contributed to this work
*For correspondence. E-mail: [email protected], [email protected]
Keywords: Magnaporthe, conidiation, transcription factor, metabolic reprogramming, fluffy gene, melanization
Abstract The Magnaporthe oryzae genes FLB3 and FLB4, orthologues of the Aspergillus nidulans regulators of conidiation FlbC and FlbD, were inactivated. These genes encode C2H2 zinc finger and Myb-like transcription factors, respectively, in A. nidulans. Analysis of the resultant mutants demonstrated that FLB4 is essential for spore formation and that strains lacking this gene are fluffy in their colony morphology due to an inability to complete conidiophore formation. Meanwhile, FLB3 is required for normal levels of aerial mycelium formation. We identified genes dependent on both transcription factors using microarray analysis. This analysis revealed that the transcription of several genes encoding proteins implicated in sporulation in Magnaporthe oryzae and other filamentous fungi are affected by FLB3 or FLB4 inactivation. Furthermore, the microarray analysis indicates that Flb3p may effectively reprogramme the cell metabolically by repressing transcription of genes encoding biosynthetic enzymes and inducing transcription of genes encoding catabolic enzymes. Additionally, qRT-PCR was employed and showed that FLB3 and FLB4 transcripts are enriched in synchronously sporulating cultures, as were the transcripts of other genes that are necessary for normal conidiation, consistent with a role for their gene products in this process.
1
1. Introduction Sporulation (conidiation) is a critical phase in the disease cycle of the rice blast fungus Magnaporthe oryzae. The asexual spores (conidia) formed are the primary means by which rice blast disease is spread. Despite the importance of conidiation for this fungus, relatively little is understood regarding the genetic determinants of the process in comparison to the detailed knowledge of the prepenetrative growth phase. Conidiation in M. oryzae occurs when the fungus emerges from lesions, either through stomata or through tears in the cuticle (Howard and Valent, 1996). Conidia are formed from conidiogenous cells by a blastic mode of development (Cole, 1986; Howard, 1994). The conidia are arranged sympodially on the mature conidiophore, which normally bears three or more spores (Howard, 1994). Identification of the genetic determinants of conidiation has benefited from the availability of the Magnaporthe oryzae genome sequence and from large-scale mutagenesis programmes (Dean et al., 2005; Jeon et al., 2007). Two research groups have independently identified the MoHOX2/HTF1-homeobox transcription factor-encoding gene as essential for spore formation (Kim et al., 2009; Liu et al., 2010). Furthermore, the phosphotransferase-encoding gene MoYPD1 was recently also found to be somehow involved in conidiation. Loss of function mutants of MoYPD1 could not produce spores in various conditions (Jacob et al., 2015). These are the only genes identified to date whose mutation leads to a complete loss of conidiation in Magnaporthe; however, several mutants have been identified which show aberrant conidiophore or spore morphology and/or altered conidia production. The gene ACR1 was identified in a random mutagenesis project. ACR1 deletion leads to an abnormal conidiophore morphology: rather than the normal sympodial arrangement, the conidia of ∆acr1 mutants are produced in a chain and the gene was, thus, named ACR1 in view of this acropetal arrangement (Lau and Hamer, 1998; Nishimura et al., 2000). Further mutations affecting conidiation (con mutants) were identified by chemical or insertional mutagenesis (Shi and Leung, 1995; Shi et al., 1998). The ∆con5 and ∆con6 mutants are aconidial, the ∆con1 and ∆con2 strains display much reduced conidiation and produce misshapen spores, and the ∆con4 and ∆con7 mutants have abnormal spore morphology. In most cases, the CON gene affected has not yet been isolated or characterised, the exception being CON7, which was subsequently shown to encode a transcription factor which regulates the transcription of genes whose products influence the biogenesis of the M. oryzae cell wall (Odenbach et al., 2007). Further transcriptional regulators involved in sporulation include the MADS-box transcription factor MIG1. Mutants without MIG1 show reduced aerial mycelium growth and conidiation (Mehrabi et al., 2008). Recent large-scale mutagenesis programmes have identified many other sporulation mutants, however, a relationship between phenotype and implied genotype based on the recovery of DNA surrounding the insertion site has not been proved in most cases. Other genes whose products play an important role in the pre-penetrative growth phase have also been reported to be required for normal sporulation: notably the ∆mosnf1, ∆mps1 and ∆tps1 mutants all show an extreme reduction in sporulation (Foster et al., 2003; Xu et al., 1998; Yi et al., 2008). In the case of MoSNF1 and MPS1, which encode protein kinases required for normal virulence, these observations would suggest that signal transduction pathways controlling conidiation might share these components with some of the signalling pathways which regulate appressorium formation and/or function. The M. oryzae regulator of Gprotein signalling Rgs1p has recently been shown to negatively regulate conidiation via the G-subunit MagBp (Liu et al., 2007). Evidence for direct MagBp-Rgs1p interaction suggests that Rgs1p acts to accelerate the hydrolysis of GTP bound to MagBp and, therefore, regulates MagBp-dependent signalling negatively, hence leading to extreme reduction in conidiation of the ∆magB mutant and 2
the hyper-conidiation of the ∆rgs1 strain (Liu et al., 2007). The same study also revealed that a constitutively active form of the G-subunit MagAp subunit leads to reduced formation of aerial hyphae. The ultimate outputs for these signalling pathways in terms of changes in transcription are unclear. A recent study used microarray technology to identify genes up- and down-regulated during conidiation (Kim and Lee, 2012) and will serve as a useful resource for understanding conidiation in Magnaporthe. In comparison to the situation in the model ascomycetes Neurospora crassa and A. nidulans, relatively little is known about the genetic determinants of the conidiation process in M. oryzae. APSES transcriptional regulators, such as StuA in A. nidulans, are involved in conidiophore development, and loss of function mutants of MSTU1 in M. oryzae were found to produce a greatly decreased number of conidia (Nishimura et al., 2009). In the current study, we explored the consequences of the inactivation of the M. oryzae genes FLB3 and FLB4, which are predicted, based on sequence similarity, to encode the orthologues of the A. nidulans regulators of sporulation FlbCp and FlbDp. The implication of FlbDp in the conidiation-related process in A. nidulans has been elucidated previously, since the FlbD-deficient mutants were found to be abolished in conidiation. The FlbD gene was shown to be sufficient to direct conidiophore formation (Garzia et al., 2010; Wieser and Adams, 1995). Furthermore, the inactivation of FlbD revealed no alterations in the mycelial growth of A. nidulans, leading to the assumption that FlbD operates in a conidiation-specific manner. The FlbC gene, in its turn, was shown previously to apparently play a role in the transition from vegetative growth to development, regulating the time-specific expression of the conidiationrelated genes BrlA, AbaA and VosA. Thus, deletion of FlbC gene in A. nidulans resulted in a delayed conidiophore formation, a reduced expression of BrlA, AbaA and VosA, and a significantly reduced conidial germination, whereas the overexpression of the FlbC gene, on the contrary, activates the expression of conidiation-related genes and inhibits the hyphal growth (Kwon et al., 2010). We examined the role of FlbC and FlbD orthologues in M. oryzae to address the question whether A. nidulans and M. oryzae share common genetic regulators of molecular mechanism controlling asexual sporulation. The transcriptional profiling of FLB3- and FLB4-deficient mutants by microarray experiment should provide insights into the complexity of the regulation network of the conidiationrelated process by surveying the Flb3p- and Flb4p-dependent regulons. Our results indicate that M. oryzae Flb4p is essential for conidiation, while Flb3p is required for normal levels of aerial mycelium formation and, therefore, also for normal levels of sporulation. Comparison of the transcriptome of ∆flb3, ∆flb4 and wild type (WT) using microarray analysis revealed that the Flb3p and Flb4p regulons overlap significantly, but that Flb3p has a broader influence and may control a general response to nutrient limitation. The formation of aerial mycelium, together with an induction of the machinery for utilisation of five alternative carbon sources, is coupled to a reduction in the transcription of genes whose products would be expected to have biosynthetic activity.
2. Materials and Methods 2.1. Strains, materials, preparation of fungal cultures, phenotypic analysis The strains created in the present study were derived from M. oryzae WT strain 70-15 (Chao and Ellingboe, 1991), Guy11 or P1.2 (∆KU80) (Villalba et al., 2008). Fungal strains were maintained on complete medium (CM) agar plates (Talbot et al., 1993), which support good levels of sporulation. 3
We additionally used oatmeal agar (Crawford et al., 1986) and minimal media (MM) (Leung et al., 1995) supplemented with 1 % (w/v) of glucose, maltose, sucrose, fructose, glycerol, amino acids, gelatin, olive oil, cellulose, starch or xylan as a sole carbon source, respectively, to test for sporulation of mutants on different growth substrates. Standard methods for manipulation of DNA were used (Sambrook et al., 1989). Escherichia coli strain XL1-Blue (Stratagene) or TOP10 (Invitrogen) was used for bacterial transformations and for the cloning and maintenance of the constructs used. All reagents and equipment were sourced from Sigma unless otherwise stated. The growth of Magnaporthe strains for assays of sporulation, disease-related morphogenesis or extraction of RNA for microarray-based transcription analysis was performed according to methods described previously (Odenbach et al., 2007). Pathogenicity tests on roots of Oryza sativa were conducted consonant with Dufresne and Osbourn (2001) and pathogenicity towards rice plants was carried out as described before (Jacob et al., 2014; Kim et al., 2009; Zhou et al., 2009). Complete medium agar cultures were scraped using a sterile scalpel for the extraction of RNA from aerial mycelium, and ungerminated spores were processed immediately following collection from CM agar cultures for the extraction of RNA, as described previously (Odenbach et al., 2007). All experiments described were repeated at least three times. At least two independently isolated mutants for each gene disruption were analysed for all phenotypic tests.
2.2. Sequence analysis and comparisons Sequence comparisons were performed using the BLASTp interrogation (Altschul and Lipman, 1990) against the National Center for Biotechnology Information non-redundant or UniProt databases. Sequence comparisons with the Magnaporthe oryzae genome were conducted via theBroad Institute of Harvard and MIT. Domain searches were performed using the programme InterPro (accessible at http://www.ebi.ac.uk/Tools/pfa/iprscan5/). Predictions of cellular localisation were made using PSORTII (http://psort.hgc.jp/form2.html; Nakai and Kanehisa, 1992). The nuclear localisation signal (NLS) prediction was performed by cNLS Mapper (http://nls-mapper.iab.keio.ac.jp) using the cut-off score for prediction of > 3.5. The alignments with orthologous protein sequences predicted from A. nidulans were conducted using ClustalW version 2.0 (Larkin et al., 2007) considering “BLOSUM” as a cost matrix with gap open cost and gap extend cost of 10 and 0.1, respectively. Manual curation, annotation and visualisation of the sequences predicted were performed using the Geneious software package (http://www.geneious.com).
2.3. Gene inactivation and complementation constructs The primers FLB3ko-F: 5’-CGACCCTTACTCCCCCTCATCAAC-3’ and FLB3ko-R: 5’TGCAAGGCTCGTAGGGGTAGAAAA-3’ were used to generate a 1932 bp PCR product, which was ligated into the vector pGEMTeasy to generate pGEM-FLB3 for the inactivation of FLB3 gene. An MscI and AfeI double restriction of pGEM-FLB3 generated a blunt-ended vector fragment, which was then ligated to the HpaI HPT cassette from pCB1003 (Carroll et al., 1994) to give pGEM-FLB3-HPT. The latter was then cleaved with ApaI and SpeI and the purified insert ligated to a pCAMBIA0380 ApaI+SpeI fragment to create the final Agrobacterium-compatible gene inactivation vector pCAMBFLB3-KO (9611 bp). The initial gene inactivation vector for FLB4 was created by PCR amplification, resulting in a 1768 bp product from 70-15 genomic DNA of the FLB4 gene with flanking regions using 4
the primers FLB4ko-F: 5’-GCGCTTCTGCATTAACTCAACTCA-3’ and FLB4ko-R: 5’CATGGCCAAACAATAAAGGAAAAA-3’. The product was cloned into pGEMTeasy to give pGEM-FLB4, which was then cleaved with EcoRV and AfeI; the vector fragment was purified and ligated to the HpaI HPT cassette from pCB1003 to give pGEM-FLB4-HPT. The latter was then restricted with ApaI+SpeI and the purified insert was ligated to ApaI+SpeI cleaved pCAMBIA0380 to give the completed gene inactivation vector pCAMB-FLB4-KO (9840 bp). The construct pCOMP-FLB3 was generated by amplification of the WT gene locus from strain 70-15 using FLB3-comp-F: 5’-GTTAAATGGACATATCTCGTGGCA-3’ and FLB3-comp-R: 5’TTGGTATACACCTAGGCATCGCT-3’ for complementation of the FLB3-disrupted mutant. The PCR product was ligated to pGEMTeasy and then the insert excised using NotI and cloned into the vector pCAMBIA-ILV (Kramer et al., 2009) cut with PspOMI, allowing the final vector to be used for the transformation of hygromycin-resistant mutants and selecting them for resistance against chlorimuron ethyl. The primers FLB4-comp-F: 5’-GTTCCTGGCCGCTTTTTAGTCTTGTG-3’ and FLB4comp-R 5’-GCGCGTCCCTTTCCTTTGTTCTC-3’ were used to amplify a blunt-ended (Phusion polymerase generated) 3545 bp product, which was restricted directly with ApaI+SpeI and ligated into pCAMBIABAR (Kramer et al., 2009) and transformed into the ∆flb4 mutant for complementation of the ∆flb4 mutant with its native promoter-gene-terminator. The plasmids pFLB3-GFP and pFLB4-GFP were constructed for generation of GFP fusion proteins. pFLB3-GFP was made by amplification of the FLB3 promoter and ORF using the primers FLB3-comp-F: 5’-GTTAAATGGACATATCTCGTGGCA-3’ and FLB3BspHI-R: 5’-TCATGAAGTCAGAGTGATGGTCCTCGGAG-3’. The PCR product was first ligated into pGEMTeasy and then the insert from the resultant vector excised with ApaI and BspHI and ligated to NcoI and ApaI cleaved pAJF-GFP (Odenbach et al., 2007). The insert from the resultant vector pFLB3GFP was excised using ApaI and SpeI and ligated into the vector pCAMBIA-HPT to create pCAMBFLB3-GFP for the transformation of the WT. The 1893 bp PCR product was generated using primers FLB4-GFP-F: 5’-GTTTCCTGCACCCCTTTTCAAGTC-3’ and FLB-GFP-R: 5’CATGATGGCAGCCTGGCGGGTGAC-3’ for GFP fusion to Flb4p. The PCR product was ligated to pGEMTeasy and then excised with ApaI and BspHI. These primers remove the stop codon and add a BspHI restriction site allowing in-frame ligation to GFP using the NcoI and ApaI site in the vector pAJF-GFP. The insert from the resultant vector pFLB4-GFP was excised using ApaI and SpeI and ligated into the vector pCAMB-ILV to create pCAMB-FLB4-GFP, which could be transformed into the ∆flb4 mutant or WT strains.
2.4. Fungal transformations The completed vectors described above were transformed into the stated WT strains using Agrobacterium tumefaciens-mediated transformation (Rho et al., 2001). Selection was performed as described previously (Odenbach et al., 2007), except for sulphonylurea-resistant transformants, which were selected on MM agar using 100 µg ml-1 chlorimuron ethyl (Sigma).
2.5. Microarray based transcription analysis Microarray-based transcription profiling experiments were performed using version III Magnaporthe oryzae oligo microarrays (Agilent Technologies). The starting material for this study was derived from 5
the RNA isolated from aerial mycelium of the synchronous solid cultures of the WT 70-15 and ∆flb3- / ∆flb4-mutants, respectively. A spore suspension with a final concentration of 1x105 spores/ml was pipetted and plated evenly on CM-agar plates covered with sterile cellophane to obtain the mycelium biomass in the case of ∆flb3-mutant. An amount of 50 ml CM in a 100-ml Erlenmeyer flask was inoculated and incubated at 28 °C and 120 U/min for 3 d for the generation of synchronous aerial mycelium of the aconidial ∆flb4-mutant. Subsequently, 1 ml of the resulting culture was plated onto a surface of CM coated with sterile cellophane. After 5 d of incubation, the RNA isolation and quality control were performed, as described previously (Odenbach et al., 2007). All procedures regarding the microarray analysis were carried out consonant with the manufacturer’s recommendations. Four biological replications were performed, each time with a reciprocal labelling experiment (“dye-swap”). Data normalization and statistical analyses were performed using Bioconductor R package Limma (Smyth, 2004). Raw signals were background corrected by subtracting the local spot background. We applied two normalization steps: firstly, within the array, using global loess normalization, and secondly, between the arrays, using the normalizing method “Aquantile”. Differentially expressed genes were obtained by fitting a linear model for each gene into the series of arrays and applying empirical Bayes. The p-values for genes differentially regulated were adjusted using the Benjamini–Hochberg procedure for false discovery rate control (Benjamini and Hochberg, 1995). The results were filtered by cut-off values for the signal intensity A > 28, the logarithmic fold change > 1 and the p-value < 0.05. We have deposited the raw data at GEO under SuperSeries accession number GSE38329. All details are compliant with MIAME.
2.6. RNA isolation, cDNA amplification and qRT-PCR analysis The RNA template used for the cDNA synthesis was extracted from 70-15 mycelium grown either from point inoculated cultures or from cultures which were created by spreading a spore suspension over the plate surface. The RNA isolation and qRT-PCR analysis were conducted as described previously (Odenbach et al., 2007). The qRT-PCR reactions were performed using three independent biological replicates. The Pfaffl analysis model was applied (Pfaffl, 2001), under consideration of EFA1 (Elongation factor 1-alpha encoding gene MGG_03641) as a reference gene, for the quantification of qRT-PCR transcription analysis.
3. Results 3.1. Identification of potential conidiation-related genes of Magnaporthe oryzae The sequences of the A. nidulans transcription factors FlbCp (UniProt: O74256_EMEND) (Wieser et al., 1994) and FlbDp (UniProt: Q00658_EMEND) (Wieser and Adams, 1995) were used to interrogate the M. oryzae genome sequence (Dean et al., 2005) for the presence of potential orthologues. A predicted protein with significant similarity to the query sequence was identified in each case. In the case of FlbCp, the similarity between these likely orthologues is confined to the C-terminal half of the proteins, where 121 out of the last 193 amino acids are identical (63 %). The similarity between A. nidulans FlbDp and its M. oryzae orthologue Flb4p is limited to the N-terminus, where the two proteins have 56 out of the last 118 of amino acids in common (~47 %). In the case of both these pairs of orthologous proteins, the similarity is largely confined to the region where the predicted DNA 6
binding domains are located. The Magnaporthe genes which encode these predicted orthologues of A. nidulans – FlbCp and FlbDp – were named FLB3 (Broad ID: MGG_04699) and FLB4 (Broad ID: MGG_06898), respectively. Sequence analyses were performed to compare the domain structure of the potential M. oryzae proteins identified with their corresponding orthologues in A. nidulans (Fig. S1). FLB3 is predicted to encode a protein with 392 amino acids which has two C2H2 zinc finger domains (PFAM ID: PF00096) at the C-terminal end of the protein (amino acids 324-346 and 352376). Two C-terminally located C2H2 zinc finger domains are also present in A. nidulans FlbCp. The predicted M. oryzae Flb3p protein has one potential NLS at position 369-379 (LRRHRKVHKGD) and one identical sequence is also present at a similar location in A. nidulans FlbCp. The FLB4 gene is predicted to encode a protein with 321 amino acids. Two closely spaced Myb-like potential DNAbinding domains (PFAM ID: PF00249) are located between amino acids 14-65 and 68-116. A very similar N-terminal arrangement of Myb-like domains is present in A. nidulans FlbDp as well as in the N. crassa orthologue RCA-1p. The residues 117-141 within M. oryzae Flb4p are predicted to form a coiled region. A potential bipartite NLS (RQNKLKMAEKKQMAQRKAEQDAKAAAKMASI) is present between amino acids 113 and 143.
3.2. FLB3 and FLB4 gene products localise to the nucleus, validating their predicted role of transcription factors In frame fusions of the FLB3 and FLB4 open reading frames (ORFs) to the GFP-encoding eGFP gene were used to demonstrate that the fusion proteins localise to the nucleus. The Flb4-GFP fusion protein showed, on average, a very faint fluorescence in spores and mycelium; however, it was found to be nuclear localised in both cell types, as was the Flb3p-GFP fusion protein (Fig. 1). The low abundance of the Flb4-GFP fusion protein may be due to the very low transcript abundance we have recorded for FLB4 within mycelium and spores (data not shown). Together, these analyses suggested that M. oryzae Flb3p and Flb4p might be transcription factors and orthologues of the A. nidulans regulators of sporulation FlbCp and FlbDp, respectively.
3.3. FLB3 and FLB4 encode products which regulate discrete steps in asexual spore formation and control melanin biosynthesis in M. oryzae We created ∆flb3 and ∆flb4 mutants using targeted gene disruption to investigate whether the M. oryzae proteins Flb3p and Flb4p have a role in the regulation of sporulation in a similar manner to FlbCp and FlbDp of A. nidulans investigated previously. Several transformants were recovered for both genes. Evaluation by southern blot analysis indicated that the gene had been successfully replaced with the hygromycin phosphotransferase-encoding gene (HPT) by homologous recombination in each case (Fig. S2). The conidiation of the ∆flb3 and ∆flb4 mutants thus generated was compared to that of the WT strain. This analysis revealed the ∆flb4 mutant to be aconidial on all media we tested, while the ∆flb3 mutant showed reduced sporulation on CM agar plates (28 ± 4 % of that of the WT). Conidiophores or even conidia-like structures was not detectable in ∆flb4 mutant. The amount of aerial mycelium formed by the ∆flb4 mutant was comparable to that of the WT; however, the culture is white and fluffy, as the aerial mycelium formed fails to differentiate (Fig. 2A). A drastic reduction in the production of aerial mycelium was observed with the ∆flb3 mutant, giving the culture a flat appearance (Fig. 2A). Fewer conidiophores are formed by the ∆flb3 mutant 7
compared to the WT strain, however, displaying a normal morphology (Fig. 2B). Pigmentation of the mutants cultivated on CM agar medium also differed: the ∆flb3 strains being darker than WT, while the ∆flb4 strains lacked pigment and were white or only showed pigmented sectors during the initial growth phase (Fig. 2C). It was not possible to assess the virulence of the ∆flb4 mutant by normal means, as infection in Magnaporthe is dependent on appressoria, which are formed after conidia have germinated. Nevertheless, we attempted to see whether we could infect rice leaves by placing mycelial blocks onto the leaf surface. It has been reported that appressoria can arise directly from the mycelial fragments in the case of the ∆cos1 mutant of M. oryzae and with certain WT strains (Kim et al., 2009; Zhou et al., 2009). By using this method, lesion formation from inoculations with mycelium could be found using the WT strain and ∆flb4, but was not observed using the ∆flb4 strain. We also didn’t find any appressoria-like structures at the hyphae tips of the mycelium used for the plant assays. Magnaporthe strains are also able to infect the roots of their hosts inoculated with mycelial fragments (Sesma and Osbourn, 2004). Therefore, we also tested whether the ∆flb4 strain was affected in its ability to infect roots. This analysis showed that the inoculations with the mutant gave rise to lesions like those of the WT (Fig. S3). Although the mechanisms by which roots and leaves are infected differ (Sesma and Osbourn, 2004), these results are consistent with the view that Flb4p may be a sporulation-specific regulatory factor. The ∆flb3 mutants, meanwhile, were unaffected in their virulence to susceptible rice cultivar CO-39 or to the barley cultivar “golden promise”, and lesions on leaves (data not shown) or roots (Fig. S3) were like those of the WT control. We reintroduced the FLB3 and FLB4 genes under the control of their native promoters and transcriptional termination regions into the respective mutants to prove that the defects we had observed were due to the inactivation of the target genes and not to some unrelated mutation. In the case of the ∆flb3 mutant, ∆flb3/FLB3 complemented strains were created by ectopic integration of the FLB3 native promoter-ORF-terminator. These strains were restored to WT levels in their production of conidia and aerial mycelium, indicating that the phenotypes we had observed in ∆flb3 strains were, in fact, due to FLB3 inactivation (data not shown). Similarly, we could restore spore formation and pigmentation to the ∆flb4 mutant by reintroduction of the FLB4 gene with its native promoter or with the Flb4-GFP fusion protein, although in both cases, WT levels of sporulation were not seen (data not shown). To prove that the defects we had seen were not strain specific, we also performed the FLB4 disruption experiment in two further WT strains, Guy11 and P1.2 (Villalba et al., 2008), and recovered multiple FLB4-disrupted strains which were verified by southern blotting. As expected, the corresponding mutants were aconidial, fluffy and displayed markedly reduced pigmentation in every case. Together, our experiments provide convincing evidence that the product of FLB4 is essential for sporulation and that the FLB3 gene product is required for production of normal levels of aerial mycelium, conidiophores and, therefore, also for normal levels of conidiation in M. oryzae.
3.4. Identification of potential Flb3p- and Flb4p-regulated genes using microarray-based transcriptome analysis Because FLB3 and FLB4 are predicted to encode transcription factors important for conidiation in M. oryzae, it was of interest to identify genes whose transcription is dependent on these proteins. Transcript abundance within total RNA isolated from mycelium scraped from the surface of CM agar cultures of the mutants and the WT (70-15) was compared using microarray experiments (summarised in Fig. 3 and shown in detail in Table S1). In the case of the ∆flb3 mutant, 474 genes 8
were found whose transcript abundance was significantly reduced. In order to extract the maximal meaning from the results, we then manually annotated these genes by assigning a function based on the presence of conserved functional domains and/or the best meaningful match to the predicted protein using BLASTp interrogation against the UniProt database (Jain et al., 2009). We found this manual method of classifying the genes identified consonant with function to be superior to our attempts at automated annotation using GO terms. Our results revealed that there were several conidiation-related genes among the genes whose transcript abundance was decreased by FLB3 inactivation, suggesting their transcriptional regulation by an FLB3-encoded transcription factor. This gene set includes, for instance, MPG1 (MGG_10315), which encodes a hydrophobin associated with aerial mycelium and known to be required for normal sporulation and virulence in M. oryzae (Talbot et al., 1993; Talbot et al., 1996). The set also includes the ACR1 (MGG_09847) gene, already discussed in the introduction, orthologues of the N. crassa CON6 and CON8 (MGG_02246; MGG_00513) genes, which are described as conidiation-specific in this species (Berlin and Yanofsky, 1985; Roberts and Yanofsky, 1989), and the MoFLP1 (MGG_02884) gene, whose inactivation leads to a mutant phenotype similar to that observed in the case of the FLB3-disruption mutant (Liu et al., 2009). Another gene whose transcript showed reduced abundance consistently in the ∆flb3 mutant is predicted to encode the orthologue of the A. nidulans PhiA protein (MGG_00081), which is essential for phialide development in this species (Melin et al., 2003). Additionally, many genes had predicted products which were involved in the utilisation of diverse carbon sources (especially cellulose and fatty acids) or whose transporters of varied function showed reduced transcript abundance. Several of the predicted transcriptional regulators were also among the predicted products of genes whose transcript showed a decreased abundance in the ∆flb3 mutants; however, the transcription of the FLB4 gene was not affected by FLB3-inactivation. Interestingly, the gene TRA1 (MGG_10197), previously shown to encode a transcription factor required for normal adhesion, germination, appressorium formation and virulence, was also among the genes with reduced transcript abundance (Breth et al., 2013). At the same time, the microarray analysis revealed 315 genes with reproducibly increased transcriptional level in aerial mycelium of the FLB3-disruption mutant. Interestingly, factors with biosynthetic (including those involved in synthesis of fatty acids, amino acids and melanin) rather than catabolic activity dominated among the predicted products encoded by genes with increased transcript abundance in the ∆flb3 mutant. In the case of the ∆flb4 mutant, 107 genes were found to be significantly down-regulated and 40 genes displayed a reproducibly increased abundance. More than half of the genes significantly downregulated also displayed decreased transcript abundance in the ∆flb3 mutant, including the MoFLP1 and MPG1 genes mentioned above and the CHS7 gene encoding class VII chitin synthase, previously shown to be involved in appressorium development and virulence in M. oryzae (Odenbach et al., 2009). Notable exceptions were the melanin biosynthetic genes BUF1 (MGG_02252) and ALB1 (MGG_07219) (Chumley and Valent, 1990; Oh et al., 2008) which show decreased transcript abundance in the ∆flb4 mutant and increased abundance in the ∆flb3 strain, providing, at the same time, a likely explanation for the increased pigmentation of ∆flb3 and the decreased pigmentation of ∆flb4 strains. The predicted products of the genes RGS7 (MGG_11693) and RGS8 (MGG_13926) harbouring the “regulator of G protein signalling” domain (PS50132) exhibited a dramatic reduction in transcript abundance in the ∆flb4 mutant, whereas in the case of the ∆flb3 mutant, only RGS8 revealed a decreased amount of the corresponding transcripts. However, mutants lacking these factors were previously shown to be unaffected in asexual spore formation (Zhang et al., 2011). 9
3.5. Role of the FLB3 gene product during growth on different carbon sources As we had observed that a large set of genes encoding carbohydrate-active enzymes showed reduced transcript abundance in the ∆flb3 strain, we tested the growth and sporulation of this mutant on different carbon sources including glucose, fructose, maltose, gelatin, amino acids and olive oil (Fig. 4). The outcome of these tests was the observation that although sporulation of the mutant was reduced on most carbon sources tested, the level of sporulation was carbon sourcedependent, as more spores were produced compared to the WT when cultivating on MM maltose. Notably, a well conserved maltose transporter is the product of one gene whose transcript abundance was found to be increased in ∆flb3, providing a possible explanation for the increased sporulation of this strain on MM maltose. The results from these growth tests also imply that media that support low growth of the fungus are also not good substrates for spore production, such as olive oil. Olive oil leads to sparse growth of the fungal culture and to very low spore production, i.e. on MM with olive oil (data not shown) or on CM supplemented with olive oil and without glucose. This could presumably indicate that a certain level of biomass must be accumulated before sporulation is induced and/or that nutrient availability locally is sensed to set the sporulation process in motion and that this critical level is reached much later for a sparsely growing Magnaporthe colony. Conceivable triggers for sporulation within the lesions of the rice leaf might be multiple factors, for example, nutrient status, light (Lee et al., 2006), desiccation or oxidative stress (among others). Except for light, we have found no evidence that any of these acts induce sporulation in axenic cultures (data not shown).
3.6. Analysis of transcription of FLB3, FLB4 and genes identified as being dependent on their products during conidiation We looked at the transcription of the genes in the WT strain 70-15 studied during growth on CM agar plates under two different conditions: point-inoculated cultures, where the oldest mycelium is towards the centre of the plate, and what we have termed “synchronously conidiating cultures”, which were created by spreading a spore suspension over the whole of the plate. Synchronously conidiating cultures produced at least twofold more conidia after 9 d (Fig. 5A). Therefore, it is logical to conclude that the proportion of the colony engaged in sporulation is probably higher in synchronously conidiating cultures than in point-inoculated cultures. We believe that a comparison of transcript abundance between these two different conditions would be a useful means to assess whether a specific gene was induced during sporulation, as such genes would be expected to show a higher transcript abundance in synchronously conidiating cultures than in point-inoculated cultures. Indeed, we found that FLB3, FLB4, ACR1, MPG1, MoFLP1 and others showed higher transcript abundance in synchronously conidiating cultures (Fig. 5B). Meanwhile the transcript of a gene UVI1 (MGG_02647) that we have shown previously to be dispensable for spore formation (data not shown) displays no differential transcript abundance. Based on our results, we propose that synchronously conidiating cultures versus point-inoculated cultures might be a simple and effective means to search for sporulation-related factors by transcription profiling.
10
3.7. Comparison between microarray analysis investigating conidiation A study was published recently which examined transcriptional changes associated with sporulation from the KJ201 strain (Kim and Lee, 2012). The study clearly demonstrates that the transcription of both FLB3 and FLB4 is induced during sporulation, as we have proposed in the previous section. The study also compared microarray results between the WT and a HOX2-deleted strain which forms increased numbers of conidiophores, but no spores, and revealed that the transcription of both FLB3 and FLB4 is Hox2p-dependent. There are, therefore, several good reasons to compare the results of the current study with this published work. The transcription of those genes up-regulated during sporulation and, at the same time, showing decreased transcript abundance in our mutants might be considered especially good candidates for genes whose products are specifically involved in the sporulation process. Meanwhile, those genes whose transcripts show only decreased abundance in the ∆flb3, ∆flb4 or ∆hox2 mutants might represent those whose products have other roles during development (non-sporulation related activities of the transcription factors predicted). Additionally, the results of the work published to identify genes involved in sporulation (as the authors state) will include some transcriptional changes unrelated to sporulation, but related to the difference in age between the cultures used. Furthermore, genes with a low level of expression in more than one mutant (e.g. ∆hox2 and ∆flb4, whose phenotypes are most similar) and normally induced are good candidates for conserved sporulation factors. We performed a comparison of differentially expressed genes using Venn diagrams to identify sets of genes shared across the conditions mentioned previously and, thus, to deduce potential candidate genes for association with the conidiation process. General comparisons across the arrays are summarised in Table 1, and some of the pathogenicity- and conidiation-relevant genes known from previous studies and being shared across compared microarray results are visualized using Venn diagrams (Fig. S4A, B). The differentially regulated genes in common between different experiments are listed in Table S1, which also provides a list of potential candidate genes for further investigation, based on their hypothesized involvement in the conidiation process. We also performed a manual annotation of the results of Kim and Lee (2012), similar to that we made for our results (Table S2), to provide an objective comparison. In the case of the ∆flb4 mutant, a significant overlap between the FLB3 and FLB4 regulons was apparent, as one would anticipate based on the phenotypes of the respective mutants (~52 % of genes with reduced transcript abundance in the ∆flb4 mutant also exhibited reduced transcript abundance in the ∆flb3 mutant). Genes displaying a reduced transcript abundance in ∆flb3, but not in ∆flb4 will, therefore, be included, however, these will not be limited to those whose products are specifically involved in the initiation of aerial mycelium formation and not in later stages of the sporulation process. Our analysis of the FLB3- and FLB4-disruption stains had suggested a close link between the regulation of melanin biosynthesis and melanisation; however, a comparison with published microarrays makes it clear that this is not the case, and we propose that regulation of melanin biosynthesis may be a role different to and independent of the role of the Flb3p and Flb4p transcription factors in the sporulation process.
4. Discussion Relatively sparse information exists to date on the molecular basis of conidiation in M. oryzae, even though this process plays a critical role in the infection cycle. Thus, investigation and deepening our understanding regarding this process could provide a very important means to control the disease in 11
the field. Decoding of the signaling pathways involved in conidiation represents an effective approach to identify the genetic determinants of conidiation-associated processes, encompassing both the initiation and late phases of the conidiation. At the same time, much more extensive and detailed information exists on molecular mechanisms regarding the conidiation process in A. nidulans and N. crassa. The objectives of this study were to characterize fluffy gene homologues involved in conidiogenesis and their mechanism of action in M. oryzae. We characterized two fluffy gene homologues in M. oryzae and found that FlbDp and FlbCp are conserved regulators for conidiogenesis in A. nidulans and M. oryzae. Thus, disruption of the FlbD coding sequence in M. oryzae resulted in mutants lacking the conidiophore differentiation and the formation of spores, whereas the inactivation of the FlbC gene led to mutants unable to produce the intact conidiophores. Using the basic bioinformatic analysis, we were, furthermore, able to identify the other putative homologous genes in M. oryzae as known and described factors also involved in the conidiation process in A. nidulans and/or N. crassa, but with some exceptions. Hence, the observation that the gene encoding the transcription factor BrlAp, the unit of functional conidiation triad BrlAp-AbaApWetAp, playing a critical role in the activiation of sporulation pathway, is absent in the genome of M. oryzae is very interesting. Therefore, we suggest that, in the case of Magnaporthe, there must be other/additional mechanisms for the regulation of sporulation that might differ from the conserved regulation mechanisms. Although Kim et Al. have postulated in their study that the gene with the annotation number MGG_00501 encodes the BrlAp factor, whose expression would fit the Aspergillus nidulans hypothetical model very well, it is questionable whether it is indeed the gene, due to a weak similarity to its A. nidulans homologue (Liu et al., 2010). According to the A. nidulans model, FlbAp, FlbBp, FlbCp, FlbDp and FlbEp act as early regulators that activate the downstream operating central regulatory pathway in response to the product of FluG activity (Kim et al., 2009). The central genetic pathway of conidiation, in its turn, is composed of BrlAp, AbaAp and WetAp, coordinately regulating the order of gene activation during conidiophore development and spore maturation (Adams et al., 1998). Another example of the existence of the discrepancy concerning the conservation of regulation mechanisms of conidiation is provided by the VosA gene, previously functionally characterized in M. oryzae (Kim et al., 2014). The M. oryzae ΔvosA mutant has no defects in conidiation, although the A. nidulans VosA was previously shown to play a crucial role as a key regulator of the sporulation process (Ni and Yu, 2010). This observation provides the next evidence that gene pathways regulating conidiation may differ between fungal species and may derive from new mechanisms of gene regulation, rather than relying on changes concerning the biochemical function of the corresponding protein. Further investigation is necessary to define the genetic pathway and molecular mechanisms controlling conidiation in M. oryzae, and the understanding of the evolutionary-driven impact on the dynamics of developmental pathways is also essential for our understanding regarding the evolution of fungal adaptation concerning the pathogenicity-related processes. A microarray analysis was performed, based on the gene expression analysis of ∆flb3 and ∆flb4 mutants relative to that of the WT (70-15), to clarify the situation regarding the gene regulation of sporulation-associated processes in M. oryzae. Following this microarray analysis, we could reveal some useful first clues as to how the predicted products encoded by FLB3 and FLB4 may act to control sporulation. In line with their different roles in the process, some of the genes dependent on these factors were found to be in common, but also some of them were identified as unique to each transcription factor. The influence of Flb3p would generally seem to be broader. Flb3p obviously acts before Flb4p in the steps required to ultimately produce spores. Our results suggest that Flb4p function may be limited to the final steps in the 12
formation of conidiophores. Because aerial mycelium formation is reduced in the FLB3-disruption mutant, all subsequent steps in the sporulation process are also reduced and some degree of overlap in the microarray result was to be expected. Because the transcription of genes whose products influence carbohydrate metabolism was largely unaffected by the inactivation of FLB4, our results suggest that the influence of FLB3 on the transcription of such genes might be a function of Flb3p in addition to its role in aerial mycelium formation. In fact, it is logical to expect that the fungus would have some factors linking aerial mycelium formation (and ultimately spore formation) to nutrient status. The situation in Magnaporthe appears to be complex, as spore formation in axenic culture does not seem to be induced explicitly by starvation, similar to other fungi, and we believe that in its native environment, another factor (as yet unclear) triggers the sporulation of the fungus within lesions. Our results suggest that Flb3p may couple the initiation of aerial mycelium formation to the production of different carbohydrate-active enzymes: two logical responses to a limitation in preferred carbon sources. The results achieved in the current study allow us to draw up a hypothetical model of how the regulatory mechanism of conidiation could function in the case of M. oryzae (see Fig. 6).
Concluding remarks It would seem clear that Flb3p and Flb4p are responsible for transcriptional changes underlying the sporulation process. Flb3p appears to be implicated in the initiation of aerial mycelium formation and may also act to induce the production of factors required for the utilisation of alternative carbon sources. Flb4p may act later in sporulation and affects mainly conidiophore formation: no conidiophores or spores are formed in the absence of the protein. Melanisation may be repressed by the Flb3p function, whereas melanisation may be induced by the Flb4p function: mutants lacking FLB4 are almost completely non-pigmented. Some of the influences of these factors may operate indirectly, for example, through the control of transcription of other regulators, such as Acr1p. The roles of Flb3p and Flb4p proposed in our study are summarised in Fig. 6. The questions now are: what exactly is the timing of these transcriptional changes and what other factors are responsible for setting them in motion? In order to address these questions, we perhaps need to better understand the state of the fungal culture over time. At what point does aerial mycelium formation begin and is this simultaneous with the first appearance of conidiophores? What proportion of the aerial parts of the fungus on a solid medium are conidiophores and does this vary with time? Can we physically, genetically or pharmacologically uncouple aerial mycelium formation from sporulation (a prerequisite for differentiating between genes acting specifically on one process)? What genes are really controlled directly by the transcription factors studied to date? The current study shows the power, but also the limitations of microarray analysis. Transcriptional profiling over a time course could shed some light on the timing of events, but clearly, addressing the issues raised will require additional technologies/approaches (e.g. RNA-Seq). These are challenging questions, not least because, in contrast to the spore itself, the fungal culture is a complex and heterogeneous biomass. A final point worth some reflection is also how far can we extrapolate from studies of the fungal grown in axenic culture to the sporulation of the fungus where it really matters: in the field?
13
References Adams, T.H., Wieser, J.K., Yu, J.H., 1998. Asexual sporulation in Aspergillus nidulans. Microbiol. Mol. Biol. Rev. 62, 35–54. Altschul, S.F., Lipman, D.J., 1990. Protein database searches for multiple alignments. Proc. Natl. Acad. Sci. 87, 5509–5513. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Royal Stat. Soc. Ser. B. 57, 289–300. Berlin, V., Yanofsky, C., 1985. Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa. Mol. Cell Biol. 5, 849–855. Breth, B., Odenbach, D., Yemelin, A., Schlinck, N., Schröder, M., Bode, M., Antelo, L., Andresen, K., Thines, E., Foster, A.J., 2013. The role of the Tra1p transcription factor of Magnaporthe oryzae in spore adhesion and pathogenic development. Fungal Genet. Biol. 57, 11–22. Carroll, A., Sweigard, J., Valent, B., 1994. Improved vectors for selecting resistance to hygromycin. Fungal Genet. Newsl. 41, 22. Chao, C.C.T., Ellingboe, A.H., 1991. Selection for mating competence in Magnaporthe oryzae pathogenic to rice. Can. J. Bot. 69, 2130–2134. Chumley, F.G.,alent, B., 1990. Genetic analysis of melanin-deficient, nonpathogenic mutants of Magnaporthe grisea. Mol. Plant-Microbe Interact. 3, 135–143. Cole, G.T., 1986. Models of cell differentiation in conidial fungi. Microbiol. Rev. 50, 95–132. Crawford, M.S., Chumley, F.G., Weaver, C.G., Valent, B., 1986. Characterization of the heterokaryotic and vegetative diploid phases of Magnaporthe grisea. Genetics. 114, 1111–1129. Dean, R.A., Talbot, N.J., Ebbole, D.J., Farman, M.L., Mitchell, T.K., Orbach, M.J., Thon, M., Kulkarni, R., Xu, J.R., Pan, H., Read, N.D., Lee, Y.H., Carbone, I., Brown,D., Oh, Y.Y., Donofrio, N., Jeong, J.S., Soanes, D.M., Djonovic, S., Kolomiets, E., Rehmeyer, C., Li, W., Harding, M., Kim, S., Lebrun, M.H., Bohnert, H., Coughlan, S., Butler, J., Calvo, S., Ma, L.J., Nicol, R., Purcell, S., Nusbaum, C., Galagan, J.E., Birren, B.W., 2005. The genome sequence of the rice blast fungus Magnaporthe grisea. Nature, 434, 980–986. Dufresne, M., Osbourn, A.E., 2001. Definition of tissue-specific and general requirements for plant infection in a phytopathogenic fungus. Mol. Plant Microbe Interact. 14, 300–307. Foster, A.J., Jenkinson, J.M., Talbot, N.J., 2003. Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J. 22, 225–216, 235. Garzia, A., Etxebeste, O., Herrero‐García, E., Ugalde, U., Espeso, E.A. 2010. The concerted action of bZip and cMyb transcription factors FlbB and FlbD induces brlA expression and asexual development in Aspergillus nidulans. Mol. Microbiol. 75, 1314–1324. Howard, R.J., 1994. Cell biology of pathogenesis, in: Zeigler, S., Leong, S.A., Teng, P.S. (Eds.), Rice Blast Disease. CAB International, Wallingford, UK, pp. 3-22. Howard, R.J., Valent, B., 1996. Breaking and entering: Host penetration by the fungal rice blast pathogen Magnaporthe grisea. Annu. Rev. Microbiol. 50, 491–512.
14
Jacob, S., Foster, A.J., Yemelin, A., Thines, E., 2014. Histidine kinases mediate differentiation, stress response, and pathogenicity in Magnaporthe oryzae. Microbiologyopen 3, 668–87. Jacob, S., Foster, A.J., Yemelin, A., Thines, E., 2015. High osmolarity glycerol (HOG) signaling in Magnaporthe oryzae: Identification of MoYPD1 and its role in osmoregulation, fungicide action and pathogenicity. Fungal Biol. 119, 580–594. Jain, E., Bairoch, A., Duvaud, S., Phan, I., Redaschi, N., Suzek, B. E., Martin, M. J., McGarvey P., Gasteiger, E., 2009. Infrastructure for the life sciences: design and implementation of the UniProt website. BMC Bioinformatics 10, 1. Jeon, J., Park, S.Y., Chi, M.H., Choi, J., Park, J., Rho, H.S., Kim, S., Goh, J., Yoo, S., Choi, J., Park, J.Y., Yi, M., Yang, S., Kwon, M.J., Han, S.S., Kim, B.R., Khang, C.H., Park, B., Lim, S.E., Jung, K., Kong, S., Karunakaran, M., Oh, H.S., Kim, H., Kim, S., Park, J., Kang, S., Choi, W.B., Kang, S., Lee, Y.H., 2007. Genome-wide functional analysis of pathogenicity genes in the rice blast fungus. Nat Genet. 39, 561–565. Kim, H.J., Han, J.H., Kim, K.S., Lee, Y.H., 2014. Comparative functional analysis of the velvet gene family reveals unique roles in fungal development and pathogenicity in Magnaporthe oryzae. Fungal Genet. Biol. 66, 33–43. Kim, K.S., Lee, Y.H., 2012. Gene expression profiling during conidiation in the rice blast pathogen Magnaporthe oryzae. PLoS One. 7, e43202. Kim, S., Park, S.Y., Kim, K.S., Rho, H.S., Chi, M.H., Choi, J., Park, J., Kong, S., Park, J., Goh, J., Lee, Y.H., 2009. Homeobox transcription factors are required for conidiation and appressorium development in the rice blast fungus Magnaporthe oryzae. PLoS Genet. 5:e1000757. Kosugi, S., Hasebe, M., Tomita, M., Yanagawa, H., 2009. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc. Nat. Acad. Sci. 106, 10171– 10176. Kramer, B., Thines, E. Foster, A.J., 2009. MAP kinase signaling pathway components and targets conserved between the distantly related plant pathogenic fungi Mycosphaerella graminicola and Magnaporthe grisea. Fungal Genet. Biol. 46:667 – 681. Kwon, N.J., Garzia, A., Espeso, E.A., Ugalde, U., & Yu, J.H., 2010. FlbC is a putative nuclear C2H2 transcription factor regulating development in Aspergillus nidulans. Mol. Microbial. 77, 1203–1219. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson T.J., Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. Lau, G.W., Hamer, J.E., 1998. Acropetal: a genetic locus required for conidiophore architecture and pathogenicity in the rice blast fungus. Fungal Genet. Biol. 24, 228–239. Lee, K., Singh, P., Chung, W.C., Ash, J., Kim, T.S., Hang, L., Park, S., 2006. Light regulation of asexual development in the rice blast fungus, Magnaporthe oryzae. Fungal Genet Biol. 43, 694–706. Leung, H., Loomis, P., Pall, M.L., 1995. Transformation of Magnaporthe oryzae to phosphinothricin resistance using the bar gene from Streptomyces hygroscopicus. Fungal Genet. Newsl. 42, 41–43. Liu, H., Suresh, A., Willard, F.S., Siderovski, D.P., Lu, S., Naqvi, N.I., 2007. Rgs1 regulates multiple Gα subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism. EMBO J. 26, 690–700.
15
Liu, T.B., Chen, G.Q., Min, H., Lin, F.C., 2009. MoFLP1, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in Magnaporthe oryzae. JZhejiang Univ. Sci. B 10, 434–44. Liu, W., Xie, S., Zhao, X., Chen, X., Zheng, W., Lu, G., Xu, J-R., Wang, Z., 2010. A homeobox gene is essential for conidiogenesis of the rice blast fungus Magnaporthe oryzae. Mol. Plant Microbe Interact. 23, 366–375. Mehrabi, R., Ding, S., Xu, J.-R., 2008. MADS-box transcription factor mig1 is required for infectious growth in Magnaporthe grisea. Eukaryot. Cell 7, 791–799. Melin, P., Schnürer, J., Wagner, E.G., 2003. Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans. Fungal Genet. Biol. 40, 234–241. Nakai, K., Kanehisa, M., 1992. A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14, 897–911. Ni, M., Yu, J.H., 2007. A novel regulator couples sporogenesis and trehalose biogenesis in Aspergillus nidulans. PloS one, 2, e970. Nishimura, M., Fukada, J., Moriwaki, A., Fujikawa, T., Ohashi, M., Hibi, T., Hayashi, N., 2009. Mstu1, an APSES transcription factor, is required for appressorium-mediated infection in Magnaporthe grisea. Biosci. Biotechnol. Biochem. 73, 1779–1786. Nishimura, M., Hayashi, N., Jwa, N.S., Lau, G.W., Hamer, J.E., Hasebe, A., 2000. Insertion of the LINE retrotransposon MGL causes a conidiophore pattern mutation in Magnaporthe grisea. Mol. Plant-Microbe Interact. 8, 892–894. Odenbach, D., Breth, B., Thines, E., Weber, R.W.S., Anke, H., Foster, A.J., 2007. The transcription factor Con7p is a central regulator of infection-related morphogenesis in the rice blast fungus Magnaporthe grisea. Mol. Microbiol. 64, 293–307. Odenbach, D., Thines, E., Anke, H., Foster, A.J., 2009. The Magnaporthe grisea class VII chitin synthase is required for normal appressorial development and function. Mol. Plant Pathol. 10, 81–94. Oh, Y., Donofrio, N., Pan, H., Coughlan, S., Brown, D.E., Meng, S., Mitchell, T., Dean, R.A., 2008. Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae. Genome Biol. 9, R85. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45. Rho, H.S., Kang, S., Lee, Y.H., 2001. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol. Cells 12, 407–411. Roberts, A.N., Yanofsky, C., 1989. Genes expressed during conidiation in Neurospora crassa: characterization of con-8. Nucleic Acids Res. 17, 197–214. Sambrook J., Fritsch E.F., Maniatis T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sesma, A., Osbourn, A.E., 2004. The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi. Nature. 431, 582–586. Shi, Z., Christian, D., Leung, H., 1998. Interactions between spore morphogenetic mutations affect cell types, sporulation, and pathogenesis in Magnaporthe grisea. Mol. Plant-Microbe Interact. 11, 199–207.
16
Shi, Z., Leung, H., 1995. Genetic analysis of sporulation in Magnaporthe grisea by chemical and insertional mutagenesis. Mol. Plant-Microbe Interact. 8, 949–959. Smyth, G.K., 2004. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, 1–29. Talbot, N.J., Ebbole, D.J., Hamer, J.E., 1993. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5, 1575–1590. Talbot, N.J., Kershaw, M.J., Wakley, G.E., De Vries, O., Wessels, J., Hamer, J.E., 1996. MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. Plant Cell. 8, 985–999. Villalba, F., Collemare, J., Landraud, P., Lambou, K., Brozek, V., Cirer, B., Morin, D., Bruel, C., Beffa, R., Lebrun, M.H., 2008. Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for nonhomologous end joining. Fungal Genet. Biol. 45, 68–75. Wieser, J., Adams, T.H., 1995. flbD encodes a Myb-like DNA-binding protein that coordinates initiation of Aspergillus nidulans conidiophore development. Genes Dev. 9, 491–502. Wieser, J., Lee, B.N., Fondon, J. 3rd, Adams, T.H., 1994. Genetic requirements for initiating asexual development in Aspergillus nidulans. Curr. Genet. 27, 62–69. Xu, J.R., Staiger, C.J. Hamer, J.E., 1998. Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc. Natl. Acad. Sci. U. S. A. 95, 12713–12718. Yi, M., Park, J.H., Ahn, J.H., Lee, Y.H., 2008. MoSNF1 regulates sporulation and pathogenicity in the rice blast fungus Magnaporthe oryzae. Fungal Genet. Biol. 45, 1172–1181. Zhang, H., Tang, W., Liu, K., Huang, Q., Zhang, X., Yan, X., Chen, Y., Wang, J., Qi, Z., Wang, Z., Zheng, X., Wang, P., Zhang, Z., 2011. Eight RGS and RGS-like proteins orchestrate growth, differentiation, and pathogenicity of Magnaporthe oryzae. PLoS Pathog. 7, e1002450. Zhou, Z., Li, G., Lin, C. He, C., 2009. Conidiophore stalk-less1 encodes a putative zinc-finger protein involved in the early stage of conidiation and mycelial infection in Magnaporthe oryzae. Mol. Plant Microbe Interact. 22, 402–410.
17
Table 1: Comparison between microarray results of the current study and microarray results of Kim and Lee (2012)
during Conidiation*
in ∆flb4
in ∆hox2*
Up-regulated
Down-regulated
Up-regulated
Down-regulated
Up-regulated
Down-regulated
Down-regulated in ∆flb3 (474 genes)
18 % (85)
3.2 % (15)
0.2 % (1)
11.8 % (56)
5.7 % (27)
14 % (66)
Up-regulated in ∆flb3 (315 genes)
2.2 % (7)
15.9 % (50)
6.7 % (21)
3.2 % (10)
12.1 % (38)
2.5 % (8)
during Conidiation*
in ∆flb3
in ∆hox2*
Up-regulated
Down-regulated
Up-regulated
Down-regulated
Up-regulated
Down-regulated
Down-regulated in ∆flb4 (107 genes)
26 % (28)
3.7 % (4)
9.3 % (10)
52.3 % (56)
5.6 % (6)
15 % (16)
Up-regulated in ∆flb4 (40 genes)
0 % (0)
10 % (4)
52.5 % (21)
2.5 % (1)
17.5 % (7)
0 % (0)
Differentially expressed genes derived from the previous study by Kim and Lee (2012)
18
Figure Legends Fig. 1: Expression and cellular localisation of Flb3p::GFP and Flb4p::GFP in conidia and mycelium GFP-fusion to Flb3p and Flb4p indicates nuclear localisation of fusion proteins. Co-localisation with DAPIstained DNA was apparent for both Flb3p-GFP and Flb4-GFP proteins. Bar indicates 10 µm.
Fig. 2: Phenotypic features of the ∆flb3 and ∆flb4 mutants
A. Side view of cultures of WT (70-15) grown in CM and the ∆flb3 and ∆flb4 mutants shows that the ∆flb3 strain produces very little aerial mycelium/conidiophores, giving the culture a very flat profile. The ∆flb4 strain produces extensive tall growing aerial mycelium (without conidiophores), giving the culture a fluffy appearance. B. Conidiophores which are produced by the ∆flb3 strain resemble those of WT. Bar indicates 10 µm. C. Melanisation of the ∆flb3 strain is increased, while that of the ∆flb4 strain is decreased (most plates are totally white with rarely some pigmented regions during initial growth).
Fig. 3: Microarray results displayed by functional enrichment analysis Bar charts represent functional classification of products of the differentially regulated genes predicted to be Flb3p- and Flb4p-dependent. The products of the differentially expressed genes according to microarray analysis were manually annotated by assigning a function based on the presence of conserved functional domains and/or the best meaningful match to the predicted protein. Only the top nine enriched functionrelated terms are represented in the respective bar chart. A. Functional classification of the products of Flb3pdependent genes showing significant transcript abundance. Red bars represent significant functional categories for products of the down-regulated genes. Green bars indicate those for up-regulated genes.
B. Functional
classification of the products of Flb4p-dependent genes with significant transcript abundance. The colours of the corresponding bars are used in analogy to A. Full details of the microarray features, Broad IDs and their predicted gene products are given in Table S1.
Fig. 4: Conidiation of the FLB3-disruption mutant on varied carbon sources The number of spores per plate was assessed after incubation period of 11 d using six plates per condition; the experiment was repeated at least three times. Sporulation of the ∆flb3 mutant complemented by reintroduction of functional copy of the FLB3 gene is shown as a control, indicated as ∆flb3/FLB3. Sporulation of all strains on MM olive oil was too low to give reliable data.
Fig. 5: Sporulation of point-inoculated and synchronous cultures and use of these to analyse sporulation-related genes
A. Conidiation on two types of culture was tested: synchronous cultures which were inoculated using a spore suspension spread across the plate surface and point-inoculated cultures which were started by placing a mycelial block in the centre of the solid medium used (CM agar). Synchronous cultures produce more spores than point-inoculated cultures at later time points.
B. Quantitative reverse transcription polymerase chain
reaction (qRT-PCR) was used to assess the relative transcript abundance during sporulation using two different types of cultures of WT (70-15). Relative transcript abundance was assessed using the method of Pfaffl (2001) and EFA1 (MGG_03641) as a reference gene. Three biological replicates were performed. The transcripts of
19
FLB3 and FLB4 and the genes whose products are known mediators of sporulation-related process were significantly more abundant in the synchronous cultures.
Fig. 6: Model summarising the proposed functions for Flb3p and Flb4p Flb3p appears to be implicated in the initiation of aerial mycelium formation and may also act to induce the production of factors required for the utilisation of alternative carbon sources (both catabolic enzymes and transporters for uptake of their products). Melanisation may normally be repressed by Flb3p function. Flb4p may act later in sporulation and affects mainly conidiophore formation: no conidiophores or spores are formed in the absence of the protein. Melanisation may normally be induced by Flb4p function: mutants lacking FLB4 are almost completely non-pigmented. Some of the influences of these factors may operate indirectly, for example, through the control of transcription of other regulators, such as Acr1p.
20