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Stachyose is a preferential carbon source utilized by the rice false smut pathogen, Villosiclava virens
Physiological and Molecular Plant Pathology 96 (2016) 69e76
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Stachyose is a preferential carbon source utilized by the rice false smut pathogen, Villosiclava virens Yu-Qiu Wang a, Guo-Bang Li a, Zhi-You Gong a, Yan Li a, Fu Huang a, b, Jing Fan a, *, Wen-Ming Wang a, ** a b
Rice Research Institute & Key Laboratory for Major Crop Diseases, Sichuan Agricultural University, Chengdu 611130, China College of Agronomy & Institute of Agricultural Ecology, Sichuan Agricultural University, Chengdu 611130, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 June 2016 Received in revised form 4 September 2016 Accepted 18 September 2016 Available online 19 September 2016
Villosiclava virens is the causal pathogen of rice false smut (RFS) disease. RFS is an emerging panicle disease, concerning rice production worldwide. However, the biology and pathogenicity of V. virens are still not well-understood. In this study, we investigated the sugar preference of V. virens and the underlying mechanism on how the favored sugar promoted the fungal growth. The results demonstrated that V. virens preferentially utilized stachyose over sucrose that is previously reported to be the best carbon source for V. virens. Stachyose could promote conidia germination, hyphae elongation and mycelium growth of V. virens. Transcriptome and qRT-PCR analyses showed that genes involved in transporting carbohydrate, amino acid, inorganic ion and secondary metabolites were mostly upregulated by stachyose than by sucrose. Moreover, Uv8b_6977 encoding a putative major facilitator superfamily (MFS) transporter was highly and specifically induced by stachyose, indicating that it may be a stachyose transporter. Expression levels of several stachyose-inducible MFS genes were also highly induced in V. virens-infected rice spikelets especially when early false smut balls appeared, implicating that these genes may play important roles in the formation of false smut balls. Collectively, this work identifies stachyose as a preferential carbon source for V. virens, and provides gene candidates for investigation of the sugar utilization mechanism of V. virens. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Major facilitator superfamily Oligosaccharide Rice false smut Stachyose Ustilaginoidea virens
1. Introduction Rice false smut (RFS) disease caused by Villosiclava virens has been recorded world-wide [1e7]. This disease not only reduces rice yield but also affects grain quality [8,9]. Asian V. virens isolates can produce mycotoxins, which are poisonous to human and animals [10e12], although no detectable mycotoxins have been reported in V. virens from USA [13]. With the increasing occurrence of RFS, it has become a big concern for the rice production. V. virens is classified as an ascomycete fungus, and can produce sexual ascospores and asexual chlamydospores (namely Ustilaginoidea virens) [14]. Both ascospores and chlamydospores can
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y.-Q. Wang), 2472630931@qq. com (G.-B. Li), [email protected] (Z.-Y. Gong), [email protected] (Y. Li), [email protected] (F. Huang), [email protected] (J. Fan), [email protected] (W.-M. Wang). http://dx.doi.org/10.1016/j.pmpp.2016.09.003 0885-5765/© 2016 Elsevier Ltd. All rights reserved.
germinate and produce secondary conidia [15,16]. V. virens spores germinate at an optimal temperature between 28 C and 30 C, and potato-sucrose-agar (PSA) was the optimum medium for germination [17,18]. The mycelium growth of V. virens is very slow in all tested solid media, with the maximum growth in PSA, i.e. nearly 37 mm of colony diameter after cultured 14 days [18]. Thus, contamination is a major problem during culture. V. virens infects rice spekelets at booting stage [19,20]. The infection process generally includes two phases: (i) Epiphytic stage. At this stage, spores enter into a developing panicle and land on the surface of spikelets, then germinate followed by hyphae extending into the inner space of a spikelet via the small gap and cracks between the palea and the lemma [21,22]. In this phase, no infection sites have been detected, presumably due to its epiphytic growth [19,23e25]. (ii) Biotrophic stage. At this stage, hyphae reach the inner floral organs, primarily infect stamen filaments, and also attack stigmata and styles; lodicules and ovaries can be occasionally infected [20,22,26]. Ultimately, V. virens mycelia embrace all the floral organs and uptake abundant nutrients to form a false smut
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ball, probably through activating rice grain filling system and hijacking nutrient reservoir [19,22]. False smut ball is the only visible symptom of RFS disease, and its size is often times larger than a mature rice grain, likely requiring plenty of nutrients. However, the nutrient utilization strategy of V. virens is unclear. Rice flowers are rich in sugars, such as glucose, sucrose, oligosaccharides etc, which could be utilized by V. virens to form false smut balls. Identification of the preferential carbon source for V. virens can facilitate to uncover its pathogenesis. Previous studies suggest that sucrose is an optimum carbon source for V. virens [18,27]. To investigate whether there are any other carbon sources better than sucrose in aiding V. virens growth, we tested the effects of a list of sugars, including mono-, di-, oligo-, and polysaccharides, on V. virens growth. Then, transcriptional approaches were applied to investigate the possible mechanism on why the preferential carbon source, i.e. stachyose, facilitated V. virens growth better than sucrose. Moreover, we also examined whether stachyose promotes the growth of other filamentous phytopathogens. Our data provide a starting point to unveil the enigmatic nature that V. virens successfully colonizes rice spikelet to form the false smut ball. 2. Materials and methods 2.1. Fungal isolates and culture conditions A GFP-tagged V. virens isolate P4 [24] and two other isolates named PJ-52-2-5 and PJ78 purified from false smut balls in Ya'an (Sichuan Province, China) were used in this study. All V. virens isolates were cultured with potato-sucrose-agar (PSA) at 28 C in the dark for propagation. To identify preferential carbon source for V. virens, mycelium discs were inoculated onto Czapek agar medium (3 g NaNO3, 1 g K2HPO4$H2O, 0.5 g MgSO4$7H2O, 0.5 g KCl, 0.01 g FeSO4, 15 g agar, and H2O to a final volume of 1000 ml) supplemented with either 10 g of sucrose (Kelong, China), glucose (Solarbio, China), lactose (Solarbio, China), maltose (Solarbio, China), raffinose (Biodee, China), stachyose (Solarbio, China), or soluble starch (Kelong, China) as the sole carbon source. The mycelium area was recorded at 25 d, and the mycelia were sampled for qRT-PCR analysis. To test whether stachyose is a preferential carbon source for other microbes, three important phytopathogens including Magnaporthe oryzae (a field isolate 157 from Sichuan Province, China), Phytophthora capsici (strain 2p), and Rhizoctonia solani (a field isolate LN1 from Liaoning Province, China) were cultured in Czapek-sucrose-agar (CA) and Czapek-stachyose-agar (CStA), respectively. Mycelium area was calculated after cultured at 26 C in the dark for 10 d (M. oryzae), or at 28 C in the dark for 7 d (P. capsici and R. solani). Mycelium area was measured by Image J 1.48s [28]. Stereomicroscope Zeiss SteREO Discovery V20 (Carl Zeiss, Germany) was used to observe mycelium density. For time course expression profiling of candidate genes in response to sucrose or stachyose, P4 mycelium was first harvested from potato-sucrose-broth (PSB) medium after 7 days of culture at 28 C, 110 rpm, and then incubated in Czapek-sucrose-broth (CB) and Czapek-stachyose-broth (CStB), respectively. Mycelium was sampled at 0, 6, 12, 24, 48 h post incubation, flash frozen in liquid nitrogen and stored at 80 C until use. 2.2. Conidia germination To investigate whether stachyose affects conidia germination and hyphae elongation of V. virens, P4 was cultured in PSB at 28 C, 110 rpm for 7 d. Conidia were collected by filtration and centrifugation, and adjusted to a density of 106 conidia mL1. The conidia
were respectively incubated on CA and CStA covered with cellophane at 28 C in the dark. Condia germination was observed under a fluorescence microscope (Nikon Eclipse 80i, Nikon, Japan) at 0, 12, 24, 48, 72 h post incubation, and photos were taken with a DSFi1c camera (Nikon, Japan) connected with the fluorescence microscope. Germination rate was calculated based on at least 500 conidia. Hyphae length was measured by Image J 1.48s [28]. 2.3. Transcriptome sequencing and analysis To uncover the molecular events on how stachyose facilitates V. virens growth, RNA-seq was performed on P4 mycelium collected from CA and CStA after cultured for 28 d. Two biological replicates were included. Total RNA was extraced by using TRIzol® Reagent (Life Technologies, USA). RNA quantity and quality were determined using 2100 BioAnalyzer (Agilent Technologies Canada Inc, Mississauga, ON, Canada). RNA-seq process was described as before [19]. After filtering, clean data were mapped to the Uv8b reference genome by TopHat2 [29]. Differentially expressed genes (DEG) were obtained by cufflinks series software [30] and DEseq [31]. Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) data were used to identify differentially expressed genes (DEGs), with the criteria of absolute log2-fold change 1 and false discovery rate (FDR) 0.01. COG [32] database was used for functional classification analysis. GO [33] database was used for gene ontology analysis. 2.4. Artificial inoculation of V. virens A susceptible rice cultivar Pujiang 6 [34] was cultivated in an artificial climate chamber. P4 was cultured in PSB at 28 C, 110 rpm for 7 d, and the mixture of mycelia and conidia was homogenized and adjusted with fresh PSB to a concentration for conidia reaching 107 mL1. At the late booting stage of Pujiang 6 (5e7 d before heading), P4 inoculum was artificially injected to the developing panicles. The rice plants were kept at 26e28 C with over 85% humidity. Spikelets were sampled from the artificially inoculated or control plants at each time point (0, 1, 3, 7, 9, 11, 13, 15, 17 d post inoculation). 2.5. qRT-PCR All PCR primers are listed in S1 Table. Total RNA was extracted by using TRIzol® Reagent (Life Technologies, USA), and reverse transcriped with ReverTraAce qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan). qRT-PCR assays were performed using the QuantiFast™ SYBR® Green QPCR kit (QIAGEN, Germany). The housekeeping gene UvTub2a was used as the reference for relative expression calculation [35]. 3. Results 3.1. Stachyose is a preferred carbon source for the mycelium growth of V. virens V. virens P4 could grow in all the tested Czapek-based agar media, supplemented with sucrose, glucose, lactose, maltose, raffinose, stachyose, and soluble starch, respectively (Fig. 1A). Remarkably, stachyose-containing medium (CStA) significantly aided the growth of P4, with the mycelium area much larger than that in the media containing other sugars (Fig. 1C). For instance, the mycelium area in CStA was more than twice of that in sucrosecontaining medium (CA). To further confirm this finding, we included two other V. virens isolates for analysis. As well as P4, both PJ52-2-5 and PJ78 isolates grew better in CStA than in CA. Not only
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Fig. 1. Mycelium growth of V. virens in Czapek-agar media containing different carbon sources. A, V. virens isolate P4 was cultured at 28 C for 25 d in Czapek-agar medium supplemented with a single carbon source of sucrose, lactose, maltose, glucose, raffinose, stachyose, or soluble starch. B, Three different V. virens isolates P4, PJ52-2-5, and PJ78 were cultured at 28 C for 25 d in Czapek-based agar medium, supplemented with sucrose or stachyose, respectively. Scale bar ¼ 1 mm. C, Statistics of mycelium area from (A). D, Statistics of mycelium area from (B). The significance of mycelium area difference was calculated by Student's t-test. **p < 0.01, *p < 0.05. CA, Czapek-sucrose-agar; CStA, Czapekstachyose-agar.
the mycelium area was larger, but also the density of mycelium was higher in CStA (Fig. 1B; D). These data showed that stachyose supported better mycelium growth of V. virens than sucrose and other tested sugars. 3.2. Stachyose is better than sucrose in supporting conidia germination and hyphae elongation of V. virens V. virens conidia germination assay showed that P4 conidia germinated as early as 12 hpi on CStA; while conidia started germination at 24 hpi on CA, with germination rate eight times lower than on CStA (Fig. 2A; B). At 48 hpi, hyphae as long as 110 mm were observed on CStA, but only 10 mm on CA. At 72 hpi, hyphae with branches were extensively found on CStA, while not on CA (Fig. 2A). Collectively, compared to sucrose, stachyose accelerated conidia germination and hyphae elongation of V. virens, therefore leading to better mycelium growth (Fig. 1). 3.3. V. virens genes involved in transport are mostly up-regulated by stachysose compared to sucrose Transcriptome data showed that 20.5 and 18.7 million clean reads were obtained from CA and CStA, respectively. As a result, 89 DEGs including 58 up-regulated genes and 31 down-regulated genes were identified (Table S2). Then we randomly picked out 11 genes for qRT-PCR verification, which showed that the DEGs displayed similar trends of expression changes in both the transcriptome data and the qRT-PCR data, although the fold changes varied (Fig. 3B). This result suggests that the transcriptome data is reliable. Functional category analysis of the DEGs showed that most of them (23 out of 89) were involved in transport, including amino acid transport, carbohydrate transport, lipid transport, secondary metabolites transport and inorganic ion transport (Fig. 3A; Table S2). Seventeen of the 23 transport-associated DEGs were upregulated (Table S2). Four major facilitator superfamily (MFS) genes
were induced by at least two times, i.e. Uv8b_4811, Uv8b_4864, Uv8b_6013, and Uv8b_7624 (Table S2), which might be responsible for transporting nutrients into V. virens. Among the down-regulated genes, Uv8b_44 encoding a cell-death-inducing effector [36] was notably reduced by over 27-fold (Fig. 3B; Table S2). These data provide a list of gene candidates that may be involved in transporting stachyose or other nutrients. 3.4. Uv8b_6977 is a candidate MFS transporter specifically induced by stachyose As a first step to identify stachyose transporters, expression analysis was performed on four MFS genes up-regulated in the transcriptome data of stachyose versus sucrose (Fig. 3B), and four genes (Uv8b_1099, Uv8b_3154, Uv8b_5286, and Uv8b_6977) that were homologous to a reported oligosaccharide transporter gene Mrt in Metarhizium robertsii [37]. The qRT-PCR results showed that all the eight tested MFS genes, except Uv8b_1099 and Uv8b_3154, expressed higher in stachyose-containing medium than in other medium containing either sucrose, glucose, lactose, maltose, raffinose, or soluble starch (Fig. 4A). Noticeably, Uv8b_4811, Uv8b_4864, and Uv8b_6977 were highly induced in stachyose than in other carbon sources, with fold changes ranging from 2.5 to 30 (Fig. 4A), indicating that they are stachyose-inducible MFS genes. Furthermore, time course expression profiling of MFS genes in response to sucrose or stachyose were determined. P4 mycelium was first harvested from PSB medium after 7 days of culture, and then incubated in CB and CStB. Expression levels of Uv8b_4811 and Uv8b_6013 decreased when the mycelium was transferred from PSB to CB or CStB (Fig. 4B). Uv8b_4864 and Uv8b_5286 were only induced in mycelium transferred from PSB into CB. Interestingly, Uv8b_6977 was only induced at 12 h post transfer from PSB to CStB. Uv8b_7624 was induced in both CStB and CB, although to a lesser extent in the latter (Fig. 4B). Overall, the expression of Uv8b_6977 could be specifically induced by stachyose.
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Fig. 2. Conidia germination and hyphae elongation of V. virens in Czapek-sucrose-agar (CA) and Czapek-stachyose-agar (CstA) media. A, Conidia of a GFP-tagged V. virens isolate P4 were incubated on CA and CstA at 28 C, and observed under a flueorescence microscope at different time points post incubation. Scale bar ¼ 50 mm. B, Statistics of conidia germination rate. C, Statistics of hyphae length. Significance of differences was calculated by Student's t-test. **p < 0.01, *p < 0.05.
Fig. 3. Transcriptome analysis of V. virens in response to stachyose. A, COG (Cluster of Orthologous Groups of proteins) function classification of differentially expressed genes in V. virens cultured with stachyose, compared with sucrose. B, Validation of transcriptome data using qRT-PCR. The V. virens UvTub2a was used as a reference gene.
3.5. Expression of MFS candidates is induced in V. virens during infection of rice Time course expression patterns of putative MFS transporters were investigated in P4-inoculated rice spikelets from a susceptible
cultivar Pujiang 6. qRT-PCR demonstrated that high expression levels tended to be detected at late infection stages (including 15 dpi and 17 dpi) for all the tested genes except Uv8b_7624, which was preferentially induced at the early infection stage 3 dpi (Fig. 5). Uv8b_4811, Uv8b_4864 and Uv8b_6977 could be also induced at
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Fig. 4. Expression patterns of candidate major facilitator superfamily (MFS) transporter genes in different culture media. A, V. virens isolate P4 was cultured at 28 C for 25 d in Czapek agar medium supplemented with a single carbon source of sucrose, lactose, maltose, glucose, raffinose, stachyose, or soluble starch. Mycelium was collected for qRT-PCR analysis, and relative expression levels were calculated using UvTub2a as a reference gene. B, P4 mycelium was collected from potato-sucrose-broth (PSB) and incubated in Czapeksurose-broth (CB) and Czapek-stachyose-broth (CStB) at 28 C. Relative expression was determined at different time points post inculation with qRT-PCR, relative to the expression of reference gene UvTub2a.
early stages such as 1 dpi and 3 dpi.
V. virens.
3.6. Stachyose favors the growth of phytopathogens M. oryzae, P. capsici, and R. solani
4. Discussion
Mycelium growth of three important phytopathogens M. oryzae, P. capsici, and R. solani was monitored and compared in CA and CStA. As shown in Fig. 6, the mycelium growth was better in CStA than in CA for M. oryzae and P. capsici. Notably, the mycelium area was more than two fold in CStA than in CA for P. capsici. Obvious differences were observed for M. oryzae in both mycelium area and pigment accumulation. Although no difference was detected in mycelium area for R. solani, the mycelium density was higher in CStA than in CA (Fig. 6). These results indicate that stachyose also favors the growth of other filamentous pathogens, in addition to
Saccharides include monosaccharides (e.g. glucose and fructose), disaccharides (e.g. sucrose, lactose, and maltose), oligosaccharides (e.g. raffinose and stachyose), and polysaccharides (e.g. starch and glucogen). Sucrose or glucose has been widely used as carbon sources for culturing microbes. Previous efforts on optimizing the culture medium for V. virens have concluded that sucrose is the best carbon source, and glucose and starch can also support the growth of V. virens [18,27,38,39]. This work demonstrated that stachyose was much better than sucrose as a carbon source supporting V. virens growth at several aspects, including conidia germination, hyphae elongation, and mycelium growth.
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Fig. 5. In planta expression patterns of candidate MFS transporter genes. P4 was inoculated in the panicles of rice cultivar Pujiang 6, and spikelets were collected at different time points post inoculation (dpi) for qRT-PCR analysis. The inoculum of P4 was used as the sample at 0 dpi. Relative expression was determined with UvTub2a as the reference.
Fig. 6. Mycelium growth of several fungal pathogens in Czapek-sucrose-agar (CA) and Czapek-stachyose-agar (CstA) media. A, Magnaporthe oryzae, Phytophthora capsici, Rhizoctonia solani were cultured in CA and CStA, respectively. scale bar ¼ 200 mm. B, Mycelium area was calculated after cultured at 26 C in the dark for 10 d (M. oryzae), or at 28 C in the dark for 7 d (P. capsici and R. solani). The significance of mycelium area difference was calculated by Student's t-test. *p < 0.05.
Stachyose, belonging to the raffinose family oligosaccharide (RFO), is a tetrasaccharide and present in seeds of many crop species, such as lupin and rice [40,41]. Although stachyose is non-digestible by humans and animals due to the a-galactosidic linkages [42], it may serve as a prebiotic for some remedial bacteria in gut [43]. To our best knowledge, stachyose has not been used in culture medium for fungal phytopathogens. Our findings indicate that stachyose is so far the best carbon source for culturing V. virens. Interestingly, we also found that stachyose promoted the mycelium growth of other plant pathogens, such as M. oryzae, P. capsici, R. solani. It is suggested that stachyose may be applied for culturing different
filamentous pathogens. In the present study, transcriptome analysis revealed a possible mechanism of why stachyose promotes V. virens growth over sucrose, i.e. the presence of stachyose may induce the expression of genes involved in transporting carbohydrates, amino acids, ions, and so on. Several MFS genes were up-regulated in V. virens cultured with stachyose, compared to that with sucrose. Further transcriptional experiments identified that Uv8b_4811, Uv8b_4864 and Uv8b_6977 were highly stachyose-inducible, and Uv8b_6977 was specifically induced by stachyose, implicating its role in transporting stachyose. MFS is a large family of transporters, which
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can transfer a wide range of substrates, such as sugars and ions [44]. Metarhizium robertsii Mrt is an oligosaccharide transporter and its expression can be exclusively induced by sucrose and galactosides. Knock-out of Mrt gene can result in decreased rhizosphere competence of M. robertsii on grass roots [37]. Ustilago maydis Srt1 is a sucrose-specific transporter and is responsible for uptake of sucrose at plant/pathogen interface during infection, thus serving as an important virulence factor in U. maydis [45]. Therefore, it is interesting to investigate in future whether the stachyose-induced MFS genes, particularly Uv8b_6977, are involved in V. virens-rice interaction. V. virens attacks rice spikelets and forms false smut balls. The infection process can be generally divided into two phases, i.e. on the outer surface and in the inner space of spikelets. In our experimental conditions, no hyphae were found in the inner spikelets until around 7 dpi, which could be a time point discriminating the two phases. Expression profiling of the stachyoseinduced MFS genes showed that high expression levels were mostly detected at 15 dpi, followed by 3 dpi. This indicates that 3 dpi and 15 dpi may be critical stages for V. virens uptaking sugars. At around 3 dpi, pathogen mycelium was extensively growing on the outer surface of rice spikelets [19], representing the first phase of infection process; while white fungal mass (early false smut ball) was readily seen at 15 dpi (data not shown), suggesting that the pathogen has successfully found a nutrient source. Oligosaccharides such as stachyose and raffinose highly accumulate during the seed maturation of hybrid rice; especially, the level of stachyose is comparable to that of sucrose in the maturing seeds of Liangyou689 hybrid rice [40]. The highest expression levels of stachyoseinducible MFS genes at 15 dpi may reflect the presence of stachyose. 5. Conclusions This work identifies that stachyose is a preferential carbon source for V. virens, and supports better fungal growth than sucrose does. Stachyose also accelerates the growth of other important phytopathogens, such as M. oryzae, R. solani, and P. capsici. These findings implicate potential application of stachyose in culturing filamentous fungal pathogens. Moreover, candidate V. virens gene(s) associated with stachyose transport have been suggested; further characterization of these genes will facilitate to understand the biology and pathogenicity of V. virens. Funding information This work was supported by the National Natural Science Foundation of China (31501598). Acknowledgements We have no conflicts of interest to declare. This work was supported by the Sichuan Agricultural University Start-up packages awarded to J. Fan and W. Wang, and a grant from the National Natural Science Foundation of China (31501598) to J. Fan. We thank Prof. Y. -F. Liu (Jiangsu Academy of Agricultural Sciences, China) for kindly providing the GFP-tagged V. virens isolate P4, and Prof. D. -L. Dou (Nanjing Agricultural University, China) for providing the P. capsici strain 2p. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pmpp.2016.09.003.
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(2016), http://dx.doi.org/10.1111/1462-2920.13343. [23] J. Fan, J. Yang, Y.Q. Wang, et al., Current understanding on Villosiclava virens, a unique flower-infecting fungus causing rice false smut disease, Mol. Plant Pathol. (2015), http://dx.doi.org/10.1111/mpp.12362. [24] J. Fan, X.Y. Guo, F. Huang, et al., Epiphytic colonization of Ustilaginoidea virens on biotic and abiotic surfaces implies the widespread presence of primary inoculum for rice false smut disease, Plant Pathol. 63 (2014) 937e945. [25] E. Tanaka, T. Kumagawa, N. Ito, et al., Colonization of the vegetative stage of rice plants by the false smut fungus Villosiclava virens, as revealed by a combination of species-specific detection methods, Plant Pathol. (2016), http://dx.doi.org/10.1111/ppa.12540. [26] M. Yong, L. Fan, D. Li, et al., Villosiclava virens infects specifically rice and barley stamen filaments due to the unique host cell walls, Microsc. Res. Tech. 79 (2016) 838e844. [27] W.B. Wang, Z.Y. 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Contents lists available at ScienceDirect
Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
Stachyose is a preferential carbon source utilized by the rice false smut pathogen, Villosiclava virens Yu-Qiu Wang a, Guo-Bang Li a, Zhi-You Gong a, Yan Li a, Fu Huang a, b, Jing Fan a, *, Wen-Ming Wang a, ** a b
Rice Research Institute & Key Laboratory for Major Crop Diseases, Sichuan Agricultural University, Chengdu 611130, China College of Agronomy & Institute of Agricultural Ecology, Sichuan Agricultural University, Chengdu 611130, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 June 2016 Received in revised form 4 September 2016 Accepted 18 September 2016 Available online 19 September 2016
Villosiclava virens is the causal pathogen of rice false smut (RFS) disease. RFS is an emerging panicle disease, concerning rice production worldwide. However, the biology and pathogenicity of V. virens are still not well-understood. In this study, we investigated the sugar preference of V. virens and the underlying mechanism on how the favored sugar promoted the fungal growth. The results demonstrated that V. virens preferentially utilized stachyose over sucrose that is previously reported to be the best carbon source for V. virens. Stachyose could promote conidia germination, hyphae elongation and mycelium growth of V. virens. Transcriptome and qRT-PCR analyses showed that genes involved in transporting carbohydrate, amino acid, inorganic ion and secondary metabolites were mostly upregulated by stachyose than by sucrose. Moreover, Uv8b_6977 encoding a putative major facilitator superfamily (MFS) transporter was highly and specifically induced by stachyose, indicating that it may be a stachyose transporter. Expression levels of several stachyose-inducible MFS genes were also highly induced in V. virens-infected rice spikelets especially when early false smut balls appeared, implicating that these genes may play important roles in the formation of false smut balls. Collectively, this work identifies stachyose as a preferential carbon source for V. virens, and provides gene candidates for investigation of the sugar utilization mechanism of V. virens. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Major facilitator superfamily Oligosaccharide Rice false smut Stachyose Ustilaginoidea virens
1. Introduction Rice false smut (RFS) disease caused by Villosiclava virens has been recorded world-wide [1e7]. This disease not only reduces rice yield but also affects grain quality [8,9]. Asian V. virens isolates can produce mycotoxins, which are poisonous to human and animals [10e12], although no detectable mycotoxins have been reported in V. virens from USA [13]. With the increasing occurrence of RFS, it has become a big concern for the rice production. V. virens is classified as an ascomycete fungus, and can produce sexual ascospores and asexual chlamydospores (namely Ustilaginoidea virens) [14]. Both ascospores and chlamydospores can
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y.-Q. Wang), 2472630931@qq. com (G.-B. Li), [email protected] (Z.-Y. Gong), [email protected] (Y. Li), [email protected] (F. Huang), [email protected] (J. Fan), [email protected] (W.-M. Wang). http://dx.doi.org/10.1016/j.pmpp.2016.09.003 0885-5765/© 2016 Elsevier Ltd. All rights reserved.
germinate and produce secondary conidia [15,16]. V. virens spores germinate at an optimal temperature between 28 C and 30 C, and potato-sucrose-agar (PSA) was the optimum medium for germination [17,18]. The mycelium growth of V. virens is very slow in all tested solid media, with the maximum growth in PSA, i.e. nearly 37 mm of colony diameter after cultured 14 days [18]. Thus, contamination is a major problem during culture. V. virens infects rice spekelets at booting stage [19,20]. The infection process generally includes two phases: (i) Epiphytic stage. At this stage, spores enter into a developing panicle and land on the surface of spikelets, then germinate followed by hyphae extending into the inner space of a spikelet via the small gap and cracks between the palea and the lemma [21,22]. In this phase, no infection sites have been detected, presumably due to its epiphytic growth [19,23e25]. (ii) Biotrophic stage. At this stage, hyphae reach the inner floral organs, primarily infect stamen filaments, and also attack stigmata and styles; lodicules and ovaries can be occasionally infected [20,22,26]. Ultimately, V. virens mycelia embrace all the floral organs and uptake abundant nutrients to form a false smut
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ball, probably through activating rice grain filling system and hijacking nutrient reservoir [19,22]. False smut ball is the only visible symptom of RFS disease, and its size is often times larger than a mature rice grain, likely requiring plenty of nutrients. However, the nutrient utilization strategy of V. virens is unclear. Rice flowers are rich in sugars, such as glucose, sucrose, oligosaccharides etc, which could be utilized by V. virens to form false smut balls. Identification of the preferential carbon source for V. virens can facilitate to uncover its pathogenesis. Previous studies suggest that sucrose is an optimum carbon source for V. virens [18,27]. To investigate whether there are any other carbon sources better than sucrose in aiding V. virens growth, we tested the effects of a list of sugars, including mono-, di-, oligo-, and polysaccharides, on V. virens growth. Then, transcriptional approaches were applied to investigate the possible mechanism on why the preferential carbon source, i.e. stachyose, facilitated V. virens growth better than sucrose. Moreover, we also examined whether stachyose promotes the growth of other filamentous phytopathogens. Our data provide a starting point to unveil the enigmatic nature that V. virens successfully colonizes rice spikelet to form the false smut ball. 2. Materials and methods 2.1. Fungal isolates and culture conditions A GFP-tagged V. virens isolate P4 [24] and two other isolates named PJ-52-2-5 and PJ78 purified from false smut balls in Ya'an (Sichuan Province, China) were used in this study. All V. virens isolates were cultured with potato-sucrose-agar (PSA) at 28 C in the dark for propagation. To identify preferential carbon source for V. virens, mycelium discs were inoculated onto Czapek agar medium (3 g NaNO3, 1 g K2HPO4$H2O, 0.5 g MgSO4$7H2O, 0.5 g KCl, 0.01 g FeSO4, 15 g agar, and H2O to a final volume of 1000 ml) supplemented with either 10 g of sucrose (Kelong, China), glucose (Solarbio, China), lactose (Solarbio, China), maltose (Solarbio, China), raffinose (Biodee, China), stachyose (Solarbio, China), or soluble starch (Kelong, China) as the sole carbon source. The mycelium area was recorded at 25 d, and the mycelia were sampled for qRT-PCR analysis. To test whether stachyose is a preferential carbon source for other microbes, three important phytopathogens including Magnaporthe oryzae (a field isolate 157 from Sichuan Province, China), Phytophthora capsici (strain 2p), and Rhizoctonia solani (a field isolate LN1 from Liaoning Province, China) were cultured in Czapek-sucrose-agar (CA) and Czapek-stachyose-agar (CStA), respectively. Mycelium area was calculated after cultured at 26 C in the dark for 10 d (M. oryzae), or at 28 C in the dark for 7 d (P. capsici and R. solani). Mycelium area was measured by Image J 1.48s [28]. Stereomicroscope Zeiss SteREO Discovery V20 (Carl Zeiss, Germany) was used to observe mycelium density. For time course expression profiling of candidate genes in response to sucrose or stachyose, P4 mycelium was first harvested from potato-sucrose-broth (PSB) medium after 7 days of culture at 28 C, 110 rpm, and then incubated in Czapek-sucrose-broth (CB) and Czapek-stachyose-broth (CStB), respectively. Mycelium was sampled at 0, 6, 12, 24, 48 h post incubation, flash frozen in liquid nitrogen and stored at 80 C until use. 2.2. Conidia germination To investigate whether stachyose affects conidia germination and hyphae elongation of V. virens, P4 was cultured in PSB at 28 C, 110 rpm for 7 d. Conidia were collected by filtration and centrifugation, and adjusted to a density of 106 conidia mL1. The conidia
were respectively incubated on CA and CStA covered with cellophane at 28 C in the dark. Condia germination was observed under a fluorescence microscope (Nikon Eclipse 80i, Nikon, Japan) at 0, 12, 24, 48, 72 h post incubation, and photos were taken with a DSFi1c camera (Nikon, Japan) connected with the fluorescence microscope. Germination rate was calculated based on at least 500 conidia. Hyphae length was measured by Image J 1.48s [28]. 2.3. Transcriptome sequencing and analysis To uncover the molecular events on how stachyose facilitates V. virens growth, RNA-seq was performed on P4 mycelium collected from CA and CStA after cultured for 28 d. Two biological replicates were included. Total RNA was extraced by using TRIzol® Reagent (Life Technologies, USA). RNA quantity and quality were determined using 2100 BioAnalyzer (Agilent Technologies Canada Inc, Mississauga, ON, Canada). RNA-seq process was described as before [19]. After filtering, clean data were mapped to the Uv8b reference genome by TopHat2 [29]. Differentially expressed genes (DEG) were obtained by cufflinks series software [30] and DEseq [31]. Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) data were used to identify differentially expressed genes (DEGs), with the criteria of absolute log2-fold change 1 and false discovery rate (FDR) 0.01. COG [32] database was used for functional classification analysis. GO [33] database was used for gene ontology analysis. 2.4. Artificial inoculation of V. virens A susceptible rice cultivar Pujiang 6 [34] was cultivated in an artificial climate chamber. P4 was cultured in PSB at 28 C, 110 rpm for 7 d, and the mixture of mycelia and conidia was homogenized and adjusted with fresh PSB to a concentration for conidia reaching 107 mL1. At the late booting stage of Pujiang 6 (5e7 d before heading), P4 inoculum was artificially injected to the developing panicles. The rice plants were kept at 26e28 C with over 85% humidity. Spikelets were sampled from the artificially inoculated or control plants at each time point (0, 1, 3, 7, 9, 11, 13, 15, 17 d post inoculation). 2.5. qRT-PCR All PCR primers are listed in S1 Table. Total RNA was extracted by using TRIzol® Reagent (Life Technologies, USA), and reverse transcriped with ReverTraAce qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan). qRT-PCR assays were performed using the QuantiFast™ SYBR® Green QPCR kit (QIAGEN, Germany). The housekeeping gene UvTub2a was used as the reference for relative expression calculation [35]. 3. Results 3.1. Stachyose is a preferred carbon source for the mycelium growth of V. virens V. virens P4 could grow in all the tested Czapek-based agar media, supplemented with sucrose, glucose, lactose, maltose, raffinose, stachyose, and soluble starch, respectively (Fig. 1A). Remarkably, stachyose-containing medium (CStA) significantly aided the growth of P4, with the mycelium area much larger than that in the media containing other sugars (Fig. 1C). For instance, the mycelium area in CStA was more than twice of that in sucrosecontaining medium (CA). To further confirm this finding, we included two other V. virens isolates for analysis. As well as P4, both PJ52-2-5 and PJ78 isolates grew better in CStA than in CA. Not only
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Fig. 1. Mycelium growth of V. virens in Czapek-agar media containing different carbon sources. A, V. virens isolate P4 was cultured at 28 C for 25 d in Czapek-agar medium supplemented with a single carbon source of sucrose, lactose, maltose, glucose, raffinose, stachyose, or soluble starch. B, Three different V. virens isolates P4, PJ52-2-5, and PJ78 were cultured at 28 C for 25 d in Czapek-based agar medium, supplemented with sucrose or stachyose, respectively. Scale bar ¼ 1 mm. C, Statistics of mycelium area from (A). D, Statistics of mycelium area from (B). The significance of mycelium area difference was calculated by Student's t-test. **p < 0.01, *p < 0.05. CA, Czapek-sucrose-agar; CStA, Czapekstachyose-agar.
the mycelium area was larger, but also the density of mycelium was higher in CStA (Fig. 1B; D). These data showed that stachyose supported better mycelium growth of V. virens than sucrose and other tested sugars. 3.2. Stachyose is better than sucrose in supporting conidia germination and hyphae elongation of V. virens V. virens conidia germination assay showed that P4 conidia germinated as early as 12 hpi on CStA; while conidia started germination at 24 hpi on CA, with germination rate eight times lower than on CStA (Fig. 2A; B). At 48 hpi, hyphae as long as 110 mm were observed on CStA, but only 10 mm on CA. At 72 hpi, hyphae with branches were extensively found on CStA, while not on CA (Fig. 2A). Collectively, compared to sucrose, stachyose accelerated conidia germination and hyphae elongation of V. virens, therefore leading to better mycelium growth (Fig. 1). 3.3. V. virens genes involved in transport are mostly up-regulated by stachysose compared to sucrose Transcriptome data showed that 20.5 and 18.7 million clean reads were obtained from CA and CStA, respectively. As a result, 89 DEGs including 58 up-regulated genes and 31 down-regulated genes were identified (Table S2). Then we randomly picked out 11 genes for qRT-PCR verification, which showed that the DEGs displayed similar trends of expression changes in both the transcriptome data and the qRT-PCR data, although the fold changes varied (Fig. 3B). This result suggests that the transcriptome data is reliable. Functional category analysis of the DEGs showed that most of them (23 out of 89) were involved in transport, including amino acid transport, carbohydrate transport, lipid transport, secondary metabolites transport and inorganic ion transport (Fig. 3A; Table S2). Seventeen of the 23 transport-associated DEGs were upregulated (Table S2). Four major facilitator superfamily (MFS) genes
were induced by at least two times, i.e. Uv8b_4811, Uv8b_4864, Uv8b_6013, and Uv8b_7624 (Table S2), which might be responsible for transporting nutrients into V. virens. Among the down-regulated genes, Uv8b_44 encoding a cell-death-inducing effector [36] was notably reduced by over 27-fold (Fig. 3B; Table S2). These data provide a list of gene candidates that may be involved in transporting stachyose or other nutrients. 3.4. Uv8b_6977 is a candidate MFS transporter specifically induced by stachyose As a first step to identify stachyose transporters, expression analysis was performed on four MFS genes up-regulated in the transcriptome data of stachyose versus sucrose (Fig. 3B), and four genes (Uv8b_1099, Uv8b_3154, Uv8b_5286, and Uv8b_6977) that were homologous to a reported oligosaccharide transporter gene Mrt in Metarhizium robertsii [37]. The qRT-PCR results showed that all the eight tested MFS genes, except Uv8b_1099 and Uv8b_3154, expressed higher in stachyose-containing medium than in other medium containing either sucrose, glucose, lactose, maltose, raffinose, or soluble starch (Fig. 4A). Noticeably, Uv8b_4811, Uv8b_4864, and Uv8b_6977 were highly induced in stachyose than in other carbon sources, with fold changes ranging from 2.5 to 30 (Fig. 4A), indicating that they are stachyose-inducible MFS genes. Furthermore, time course expression profiling of MFS genes in response to sucrose or stachyose were determined. P4 mycelium was first harvested from PSB medium after 7 days of culture, and then incubated in CB and CStB. Expression levels of Uv8b_4811 and Uv8b_6013 decreased when the mycelium was transferred from PSB to CB or CStB (Fig. 4B). Uv8b_4864 and Uv8b_5286 were only induced in mycelium transferred from PSB into CB. Interestingly, Uv8b_6977 was only induced at 12 h post transfer from PSB to CStB. Uv8b_7624 was induced in both CStB and CB, although to a lesser extent in the latter (Fig. 4B). Overall, the expression of Uv8b_6977 could be specifically induced by stachyose.
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Fig. 2. Conidia germination and hyphae elongation of V. virens in Czapek-sucrose-agar (CA) and Czapek-stachyose-agar (CstA) media. A, Conidia of a GFP-tagged V. virens isolate P4 were incubated on CA and CstA at 28 C, and observed under a flueorescence microscope at different time points post incubation. Scale bar ¼ 50 mm. B, Statistics of conidia germination rate. C, Statistics of hyphae length. Significance of differences was calculated by Student's t-test. **p < 0.01, *p < 0.05.
Fig. 3. Transcriptome analysis of V. virens in response to stachyose. A, COG (Cluster of Orthologous Groups of proteins) function classification of differentially expressed genes in V. virens cultured with stachyose, compared with sucrose. B, Validation of transcriptome data using qRT-PCR. The V. virens UvTub2a was used as a reference gene.
3.5. Expression of MFS candidates is induced in V. virens during infection of rice Time course expression patterns of putative MFS transporters were investigated in P4-inoculated rice spikelets from a susceptible
cultivar Pujiang 6. qRT-PCR demonstrated that high expression levels tended to be detected at late infection stages (including 15 dpi and 17 dpi) for all the tested genes except Uv8b_7624, which was preferentially induced at the early infection stage 3 dpi (Fig. 5). Uv8b_4811, Uv8b_4864 and Uv8b_6977 could be also induced at
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Fig. 4. Expression patterns of candidate major facilitator superfamily (MFS) transporter genes in different culture media. A, V. virens isolate P4 was cultured at 28 C for 25 d in Czapek agar medium supplemented with a single carbon source of sucrose, lactose, maltose, glucose, raffinose, stachyose, or soluble starch. Mycelium was collected for qRT-PCR analysis, and relative expression levels were calculated using UvTub2a as a reference gene. B, P4 mycelium was collected from potato-sucrose-broth (PSB) and incubated in Czapeksurose-broth (CB) and Czapek-stachyose-broth (CStB) at 28 C. Relative expression was determined at different time points post inculation with qRT-PCR, relative to the expression of reference gene UvTub2a.
early stages such as 1 dpi and 3 dpi.
V. virens.
3.6. Stachyose favors the growth of phytopathogens M. oryzae, P. capsici, and R. solani
4. Discussion
Mycelium growth of three important phytopathogens M. oryzae, P. capsici, and R. solani was monitored and compared in CA and CStA. As shown in Fig. 6, the mycelium growth was better in CStA than in CA for M. oryzae and P. capsici. Notably, the mycelium area was more than two fold in CStA than in CA for P. capsici. Obvious differences were observed for M. oryzae in both mycelium area and pigment accumulation. Although no difference was detected in mycelium area for R. solani, the mycelium density was higher in CStA than in CA (Fig. 6). These results indicate that stachyose also favors the growth of other filamentous pathogens, in addition to
Saccharides include monosaccharides (e.g. glucose and fructose), disaccharides (e.g. sucrose, lactose, and maltose), oligosaccharides (e.g. raffinose and stachyose), and polysaccharides (e.g. starch and glucogen). Sucrose or glucose has been widely used as carbon sources for culturing microbes. Previous efforts on optimizing the culture medium for V. virens have concluded that sucrose is the best carbon source, and glucose and starch can also support the growth of V. virens [18,27,38,39]. This work demonstrated that stachyose was much better than sucrose as a carbon source supporting V. virens growth at several aspects, including conidia germination, hyphae elongation, and mycelium growth.
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Fig. 5. In planta expression patterns of candidate MFS transporter genes. P4 was inoculated in the panicles of rice cultivar Pujiang 6, and spikelets were collected at different time points post inoculation (dpi) for qRT-PCR analysis. The inoculum of P4 was used as the sample at 0 dpi. Relative expression was determined with UvTub2a as the reference.
Fig. 6. Mycelium growth of several fungal pathogens in Czapek-sucrose-agar (CA) and Czapek-stachyose-agar (CstA) media. A, Magnaporthe oryzae, Phytophthora capsici, Rhizoctonia solani were cultured in CA and CStA, respectively. scale bar ¼ 200 mm. B, Mycelium area was calculated after cultured at 26 C in the dark for 10 d (M. oryzae), or at 28 C in the dark for 7 d (P. capsici and R. solani). The significance of mycelium area difference was calculated by Student's t-test. *p < 0.05.
Stachyose, belonging to the raffinose family oligosaccharide (RFO), is a tetrasaccharide and present in seeds of many crop species, such as lupin and rice [40,41]. Although stachyose is non-digestible by humans and animals due to the a-galactosidic linkages [42], it may serve as a prebiotic for some remedial bacteria in gut [43]. To our best knowledge, stachyose has not been used in culture medium for fungal phytopathogens. Our findings indicate that stachyose is so far the best carbon source for culturing V. virens. Interestingly, we also found that stachyose promoted the mycelium growth of other plant pathogens, such as M. oryzae, P. capsici, R. solani. It is suggested that stachyose may be applied for culturing different
filamentous pathogens. In the present study, transcriptome analysis revealed a possible mechanism of why stachyose promotes V. virens growth over sucrose, i.e. the presence of stachyose may induce the expression of genes involved in transporting carbohydrates, amino acids, ions, and so on. Several MFS genes were up-regulated in V. virens cultured with stachyose, compared to that with sucrose. Further transcriptional experiments identified that Uv8b_4811, Uv8b_4864 and Uv8b_6977 were highly stachyose-inducible, and Uv8b_6977 was specifically induced by stachyose, implicating its role in transporting stachyose. MFS is a large family of transporters, which
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can transfer a wide range of substrates, such as sugars and ions [44]. Metarhizium robertsii Mrt is an oligosaccharide transporter and its expression can be exclusively induced by sucrose and galactosides. Knock-out of Mrt gene can result in decreased rhizosphere competence of M. robertsii on grass roots [37]. Ustilago maydis Srt1 is a sucrose-specific transporter and is responsible for uptake of sucrose at plant/pathogen interface during infection, thus serving as an important virulence factor in U. maydis [45]. Therefore, it is interesting to investigate in future whether the stachyose-induced MFS genes, particularly Uv8b_6977, are involved in V. virens-rice interaction. V. virens attacks rice spikelets and forms false smut balls. The infection process can be generally divided into two phases, i.e. on the outer surface and in the inner space of spikelets. In our experimental conditions, no hyphae were found in the inner spikelets until around 7 dpi, which could be a time point discriminating the two phases. Expression profiling of the stachyoseinduced MFS genes showed that high expression levels were mostly detected at 15 dpi, followed by 3 dpi. This indicates that 3 dpi and 15 dpi may be critical stages for V. virens uptaking sugars. At around 3 dpi, pathogen mycelium was extensively growing on the outer surface of rice spikelets [19], representing the first phase of infection process; while white fungal mass (early false smut ball) was readily seen at 15 dpi (data not shown), suggesting that the pathogen has successfully found a nutrient source. Oligosaccharides such as stachyose and raffinose highly accumulate during the seed maturation of hybrid rice; especially, the level of stachyose is comparable to that of sucrose in the maturing seeds of Liangyou689 hybrid rice [40]. The highest expression levels of stachyoseinducible MFS genes at 15 dpi may reflect the presence of stachyose. 5. Conclusions This work identifies that stachyose is a preferential carbon source for V. virens, and supports better fungal growth than sucrose does. Stachyose also accelerates the growth of other important phytopathogens, such as M. oryzae, R. solani, and P. capsici. These findings implicate potential application of stachyose in culturing filamentous fungal pathogens. Moreover, candidate V. virens gene(s) associated with stachyose transport have been suggested; further characterization of these genes will facilitate to understand the biology and pathogenicity of V. virens. Funding information This work was supported by the National Natural Science Foundation of China (31501598). Acknowledgements We have no conflicts of interest to declare. This work was supported by the Sichuan Agricultural University Start-up packages awarded to J. Fan and W. Wang, and a grant from the National Natural Science Foundation of China (31501598) to J. Fan. We thank Prof. Y. -F. Liu (Jiangsu Academy of Agricultural Sciences, China) for kindly providing the GFP-tagged V. virens isolate P4, and Prof. D. -L. Dou (Nanjing Agricultural University, China) for providing the P. capsici strain 2p. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pmpp.2016.09.003.
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