Bioinformatic analysis and functional characterization of the CFEM proteins in maize anthracnose fungus Colletotrichum graminicola

Journal of Integrative Agriculture 2020, 19(2): 541–550 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Bioinformatic analysis and functional characterization of the cfem proteins in maize anthracnose fungus Colletotrichum graminicola GONG An-dong1, 2, JING Zhong-ying1, ZHANG Kai1, TAN Qing-qun1, WANG Guo-liang1, 3, LIU Wen-de1 1

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

2

College of Life Science, Xinyang Normal University, Xinyang 464000, P.R.China Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210, USA

3

Abstract Fungal secreted proteins that contain the Common in Fungal Extracellular Membrane (CFEM) domain are important for pathogenicity. The hemibiotrophic fungus Colletotrichum graminicola causes the serious anthracnose disease of maize. In this study, we identified 24 CgCFEM proteins in the genome of C. graminicola. Phylogenic analysis revealed that these 24 proteins (CgCFEM1–24) can be divided into 2 clades based on the presence of the trans-membrane domain. Sequence alignment analysis indicated that the amino acids of the CFEM domain are highly conserved and contain 8 spaced cysteines, with the exception that CgCFEM1 and CgCFEM24 lack 1 and 2 cysteines, respectively. Ten CgCFEM proteins with a signal peptide and without the trans-membrane domain were considered as candidate effectors and, thus were selected for structural prediction and functional analyses. The CFEM domain in the candidate effectors can form a helical-basket structure homologous to the Csa2 protein in Candida albicans, which is responsible for haem acquisition and pathogenicity. Subcellular localization analysis revealed that these effectors accumulate in the cell membrane, nucleus, and cytosolic bodies. Additionally, 5 effectors, CgCFEM6, 7, 8, 9 and 15, can suppress the BAX-induced programmed cell death in Nicotiana benthamiana with or without the signal peptide. These results demonstrate that these 10 CgCFEM candidate effectors with different structures and subcellular localizations in host cells may play important roles during the pathogenic processes on maize plants. Keywords: CFEM domain, candidate effector, anthracnose disease, maize, Colletotrichum graminicola

1. Introduction

Received 26 December, 2018 Accepted 7 February, 2019 Correspondence LIU Wen-de, Tel/Fax: +86-10-62815921, E-mail: [email protected] © 2020 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.1016/S2095-3119(19)62675-4

Colletotrichum spp. as one of the top 10 fungal pathogens in plants have been served as a model system for hemibiotrophic pathogens (Dean et al. 2012). Virtually, every crop grown throughout the world is susceptible to one or more species of Colletotrichum. Among these species, Colletotrichum graminicola (Ces.) Wils., is the best characterized and most tractable species which could infect maize and cause severe anthracnose leaf blight (ALB) and

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anthracnose stalk rot (ASR) diseases in field (Schliebner et al. 2014; Torres et al. 2016; Wang et al. 2016). ALB and ASR cause a worldwide impact on maize production, for instance, the annual loss caused by this disease is up to 1 billion dollars in the USA (Miranda et al. 2017). Additionally, the incidence of maize anthracnose is likely to increase as a result of climate change and agriculture practice changes, threatening global food security. The hemibiotrophic fungal pathogen C. graminicola causes maize anthracnose disease through 3 recognizable phases including appressoria formation on the host surface prior to penetration; biotrophy, characterized by intracellular colonization of living host cells; and necrotrophy, characterized by host cell death and symptom development (Torres et al. 2016). Conidia on corn debris are the primary inoculum source of C. graminicola in fields. At proper environmental conditions, conidia germinate and form appressoria at the tips of germ tubes and generate penetration pegs which penetrate directly through the host cell wall to invade host cells (Mims and Vaillancourt 2002). The appressoria could accumulate osmolytes and generate a hydrostatic pressure, which was helpful for forcefully penetrating plant epidermis cells (Ludwig et al. 2014). Upon successful penetration of the host epidermis, biotrophic stage starts and the hyphae colonize living plant cells (Mims and Vaillancourt 2002). Biotrophy is symptomless parasitic development which is terminated at the beginning of necrotrophy. Hyphae grow quickly, breach the adjacent cells, and kill plant cells leading to visible necrosis. Biotrophic fungi affect living host cells to facilitate injection including suppressing cell death and defense responses (Doehlemann et al. 2008, 2009). In contrast, many necrotrophic fungi take advantage of plant defense responses to enhance pathogenicity, and induce host cell death by secreting phytotoxic secondary metabolites (Rolke et al. 2004; Amselem et al. 2011). Some evidence suggested that hemibiotrophic Colletotrichum spp. do both, first suppressing host cell death, then inducing cell death (Stephenson et al. 2000; Kleemann et al. 2012; Yoshino et al. 2012). Both processes appear to be regulated by secreted compounds such as effector proteins. The production of effectors is tightly regulated to provide the correct function at the appropriate time and place (Kleemann et al. 2012; Gan et al. 2013; Torres et al. 2014). Hence, identification of the repertoire of secreted proteins is of great importance for the understanding of the complex pathogenicity in hemibiotrophic pathogens. The Common in Fungal Extracellular Membrane (CFEM) domain usually encodes 60 amino acids (aa) which contain 8 characteristically spaced cysteines and it is fungal-specific (Kulkarni et al. 2003). Till now, many CFEM

domain proteins have been identified from different fungal pathogens, such as 19 proteins in Magnaporthe oryzae (Kou et al. 2017), 6 proteins in C. albicans, 13 proteins in Arthrobotrys oligospora (Zhang et al. 2015). And some of these proteins have been proved of playing essential roles in pathogenicity of these fungi such as Pth11 in M. oryzae (mediates appressorium formation) (Kou et al. 2017), Csa2 in C. albicans (utilization of hemoglobin as an iron source) (Okamoto-Shibayama et al. 2014), CfmA-C in Aspergillus fumigatus (stabilizing the cell wall) (Vaknin et al. 2014), and CgCcw14, CgMam3 in Candida glabrata (maintenance of intracellular iron content, adherence to epithelial cells and virulence) (Srivastava et al. 2014). But, the systemic identification and function analysis of CFEM-containing proteins in C. graminicola are still unknown. Here, we identified a total of 24 CgCFEM proteins in C. graminicola genome. Taking account of proteins with a signal peptide for secretion, and lack of trans-membrane domains as the major criteria for the definition of fungal effector proteins, 10 of them were fit into this category and considered as candidate effectors for functional study. These 10 candidate effectors with varied model structures, localization and suppression cell-death activity in non-host plant Nicotiana benthamiana may endow important function for pathogenicity.

2. Materials and methods 2.1. Plants and microbes Nicotiana benthamiana were grown in greenhouse at (22±1)°C with 16 h light period. C. graminicola M2 strain was cultured on PDA medium under constant light at 25°C. Agrobacterium tumefaciens was cultured in LB medium at 28°C and shaking at 200 r min–1 to produce sufficient clones.

2.2. Bioinformatic identification of CFEM-containing proteins in C. graminicola CFEM domain is unique in fungi, and it plays important roles in fungal infection and development. To identify the CFEM effector in C. graminicola, the CFEM domain-containing protein ACI1 in M. oryzae was used as query (Kulkarni and Dean 2004). The C. graminicola genome database (http:// fungi.ensembl.org/Colletotrichum_graminicola/Info/Index) was searched using Basic Local Alignment Search Tool algorithms (BLASTP) with a threshold of e-value<1e–10. All obtained proteins were examined for the presence of the CFEM domain using the PFAM tool in SMART website (http://smart.embl-heidelberg.de/). Sequences without CFEM domain were omitted manually. Additionally, the

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signal peptide and target position of these proteins were analyzed with bioinformatics tools including SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) and Target P1.1 Server (http://www.cbs.dtu.dk/services/TargetP/), respectively.

2.3. Phylogenetic analysis and multiple sequences alignment The phylogenetic tree of 24 CgCFEM proteins was constructed with the maximum likelihood method using MEGA 7.1 Program. The bootstrap analysis was conducted using the P-distance model with 500 replications. Additionally, the domain analysis of CgCFEM effectors was also drawn in our test. These domains including CFEM domain, signal peptide and trans-membrane regions were obtained from the bioinformatics analysis in SMART website and SignalP 4.1 Server, respectively. Additionally, to identify the conserved amino acid in these CgCFEM proteins, the sequence of CFEM domains were extracted and conducted for multiple sequences alignment using CLUSTAL W (Han et al. 2016). Two CFEM domains, Csa2 (XP_713316.1) in C. albicans and ACI1 (AAN64312.1) in M. oryzae were used as templates. The alignment result was modified and drawn with DNAMAN Software.

2.4. Protein model analysis of candidate CgCFEM effectors with Phyre2 Server Ten proteins containing signal peptide at N terminal and without trans-membrane regions were considered as candidate effectors. The models of these effectors were structured in Phyre2 website. Phyre2 is an available web tool to predict and analyze protein model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2 (Nasser et al. 2016). In the tests, the amino acids of 10 candidate CgCFEM effectors were uploaded and aligned in the website. The sequences were blasted with PSI-Blast for homologies, and then the Hidden Markov Model (HMM) structure was built.

2.5. Subcellular localization and hypersensitive response assay Transient expression assay of the CgCFEM were conducted by agroinfiltration method. The hypersensitive response of CgCFEM effectors was tested on N. benthamiana leaves. BAX, a death-promoting member of the Bcl-2 family proteins, which could induce programmed cell death, was used as the positive control in N. benthamiana infiltration test (Lacomme and Santa Cruz 1999). The whole open reading frame

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(referred to as FL) or the truncated coding region without the signal peptide sequence but with an engineered ATG start codon (referred to as NSP) of CgCFEM genes were cloned into pGDR transient expression vector, and infiltrated into Agrobacterium EHA105 for hypersensitive response assay (Bai et al. 2012). Agrobacteria suspensions, with OD600 (P19)=1.0, OD 600 (PGDR)=1.5, OD 600 (CgCFEM effector)=1.5, OD600 (Bax)=0.5. Each cultural suspension was mixed with equal volume of Agrobacteria P19 suspensions and cultured at dark for 2 h. Different infiltration groups were injected into tobacco leaves. After cultured for 3 days, the treated leaves were collected and detected under a Zeiss LSM710 confocal laser-scanning microscope at 559 nm excitation and 560 nm emission for subcellular localization tests. Additionally, the disease symptom in N. benthamiana leaves was taken 4–5 days post inoculation (dpi) when BAX-induced cell death was shown.

3. Results 3.1. Bioinformatics identification of CFEM protein repertoire in C. graminicola Total of 24 CFEM proteins (CgCFEM1–24) were identified in C. graminicola genome through BLASTP analysis with ACI1 from M. oryzae as query (Kulkarni and Dean 2004). The presence of CFEM domain in these proteins was further verified with SMART analysis. The protein ID number in Ensembl Fungi Database was shown in Table 1. The length of these proteins ranged from 74 to 538 aa, and CgCFEM1 is the shortest protein with 74 aa, CgCFEM17 is the largest protein with 538 aa, respectively. Among these proteins, 21 proteins, except CgCFEM11, 23 and 24, contain signal peptide in the N-terminus sequence, which were predicted as secretory protein (Table 1). A neighbor-joining phylogenetic tree was constructed based on the aa sequences of CgCFEM proteins (Fig. 1). These proteins could be clearly divided into 2 clades. One clade included 12 proteins (CgCFEM2, 5, 10, 11, 12, 13, 16, 17, 18, 19, 21 and 22), each of them contained more than 5 trans-membrane regions. In contrast, the other clade contained 12 proteins (CgCFEM1, 3, 4, 6, 7, 8, 9, 14, 15, 20, 23 and 24) that had no trans-membrane domains. Interestingly, CgCFEM23 contains 2 CFEM domains, and the position of these 2 adjacent CFEM domains is in the C-terminus which is different to other CFEM domains in N terminus. To further analyze and elucidate the conserved aa in CFEM domains, a multiple sequence alignment was conducted (Fig. 2). Two well-known CFEM containing proteins, Csa2 (XP_713316.1) in C. albicans and ACI1 (AAN64312.1) in

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Table 1 The identification of Common in Fungal Extracellular Membrane (CFEM) protein repertoire in Colletotrichum graminicola1) Name CgCFEM1 CgCFEM2 CgCFEM3 CgCFEM4 CgCFEM5 CgCFEM6 CgCFEM7 CgCFEM8 CgCFEM9 CgCFEM10 CgCFEM11 CgCFEM12 CgCFEM13 CgCFEM14 CgCFEM15 CgCFEM16 CgCFEM17 CgCFEM18 CgCFEM19 CgCFEM20 CgCFEM21 CgCFEM22 CgCFEM23 CgCFEM24

Protein ID GLRG_05629 GLRG_06380 GLRG_08687 GLRG_09687 GLRG_00781 GLRG_01006 GLRG_02462 GLRG_02673 GLRG_03926 GLRG_04780 GLRG_07733 GLRG_08253 GLRG_08904 GLRG_09163 GLRG_09340 GLRG_09400 GLRG_09453 GLRG_09694 GLRG_10780 GLRG_10834 GLRG_10885 GLRG_06414 GLRG_10361 GLRG_09189

Amino acid (aa) 74 444 284 194 457 176 214 99 156 447 422 407 461 172 165 449 538 515 473 200 428 466 420 210

TM no.

SP cleavage

mTP

SP

Other

Loc2)

RC3)

0 7 0 0 7 0 0 0 0 6 7 5 6 0 0 7 7 5 6 0 6 7 0 0

17/18 20/21 17/18 20/21 19/20 16/17 15/16 18/19 17/18 23/24 – 21/22 19/20 17/18 16/17 19/20 28/29 24/25 23/24 16/17 18/19 22/23 – –

0.016 0.056 0.058 0.032 0.059 0.061 0.043 0.032 0.047 0.027 0.037 0.114 0.344 0.030 0.101 0.023 0.038 0.044 0.037 0.063 0.037 0.019 0.236 0.162

0.96 0.862 0.842 0.932 0.974 0.825 0.812 0.922 0.850 0.977 0.200 0.793 0.741 0.916 0.902 0.974 0.956 0.953 0.974 0.672 0.955 0.970 0.054 0.096

0.109 0.039 0.088 0.066 0.017 0.120 0.165 0.124 0.129 0.024 0.887 0.034 0.013 0.147 0.062 0.038 0.017 0.020 0.021 0.316 0.042 0.046 0.745 0.730

S S S S S S S S S S – S S S S S S S S S S S – –

1 1 2 1 1 2 2 2 2 1 2 2 4 2 1 1 1 1 1 4 1 1 3 3

1)

TM, transmembrane domain; SP cleavage, cleavage site of signal peptide (SP) in the CgCFEM proteins; mTP, a mitochondrial targeting peptide prediction; Other, any other localization. 2) Loc, localization prediction. S, secretory pathway; –, no prediction. 3) RC, reliability class, ranged from 1 to 5, where 1 indicates the strongest prediction. RC is a measure of the size of the difference (diff) between the highest (winning) and the second highest output scores. There are 5 reliability classes, defined as follows: 1, diff>0.800; 2, 0.800>diff>0.600; 3, 0.600>diff>0.400; 4, 0.400>diff>0.200; 5, diff<0.200. The lower value of RC means the safer of the prediction.

M. oryzae were used as references. The length of CFEM domain is conserved in all selected proteins with about 60 aa (ranged between 54 to 71 aa). Eight conserve cysteines were found in 23 CFEM domains, which could form 4 disulfide bones to stabilize the whole protein structure of CFEM domain (Nasser et al. 2016). Exceptionally, CFEM1 (lacked the first 2 cysteines) and CFEM24 (lacked the first) missed some conserved cysteines. Additionally, relative conserved aspartic residue (position 9) was found in 17 CFEM domains, which have been proved to be important for the haem binding and extracting from haemoglobin (Nasser et al. 2016). Moreover, we also found other relative conserved aa in these CFEM domains such as proline (at position 5), valine (48, 52) and leucine (63). The different composition of CFEM domains may form various structures, and serve multiple functions during the interaction between plant and pathogens (Nasser et al. 2016). Fungal pathogen effectors play important roles during the pathogenicity, and they usually contain signal peptide, and without trans-membrane regions (Torto et al. 2003; Giraldo and Valent 2013). According to this, 10 CFEM proteins were considered as candidate effectors, and selected for further

analysis (Table 1).

3.2. Helical-basket shape detected in the model structure of CFEM domain The amino acids of 10 CFEM domains in selected candidate effectors were uploaded in Phyre2 for modeling construction and protein homology analysis. The result showed that the amino acids of these sequence have high similarity to protein Csa2 (c4y7sC in C. albicans), which is the only identified three-dimensional (3D) crystal structure of CFEM protein (Nasser et al. 2016). Each CFEM domain has been modelled with more than 97% confidence to Csa2. The structure of CFEM domain in Csa2 protein comprises 6 α-helices and 1 β-strand, which could form a helical-basket shape with an elongated N-terminal loop as handle. The model of CFEM domain in our test also contains a helicalbasket like structure similar to Csa2 (Fig. 3). Total of 6 candidate effectors (CgCFEM3, 7, 8, 9, 15 and 20) contain 4 α-helices, 3 candidate effectors (CgCFEM4, 6 and 14) contain 3 α-helices, and CgCFEM1 comprises only 2 α-helices.

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545

CgCFEM17

66

CgCFEM21 CgCFEM2

99 88

CgCFEM5

56

CgCFEM16 82

CgCFEM10

97

CgCFEM19

85

72

CgCFEM12

51

CgCFEM18

66

CgCFEM22

CgCFEM13 55

CgCFEM11

CgCFEM1 CgCFEM8 CgCFEM9 CgCFEM4 CgCFEM23

76

CgCFEM24 CgCFEM3 CgCFEM15

62 80 69

CgCFEM14 CgCFEM20 CgCFEM6 CgCFEM7

0.5

100 aa

Fig. 1 Phylogenetic analysis of the Common in Fungal Extracellular Membrane (CFEM) proteins in Colletotrichum graminicola. A neighbor-joining phylogenetic tree was constructed based on the amino acid sequences of CgCFEM effector. The numbers at nodes represent the percentage of their occurrence in 1 000 bootstrap replicates; the nodes supported more than 50% are shown in the result. The scale bar shows the number of amino acid differences per site. Green box architecture represents the length and localization of signal peptide, purple box represents the CFEM domain, blue box represents the presence of trans-membrane, respectively. The scale bar shows the length of 100 aa in the architecture.

3.3. Heterologous expression of CgCFEM effectors in N. benthamiana leaves Fungal pathogens suppress plant immunity through the secretion of numerous effector proteins. The effectors could spread into plant cells, target to different organelles, and regulate protein functions, and finally disrupt host defense signaling to increase susceptibility. Hence, effector repertoires can be key determinants of the virulence level and host range of a pathogen (Liu et al. 2014). To elucidate the localization of CgCFEM effectors in plant cells, we conducted heterologous expression analysis in N. benthamiana through agroinfiltration method with each of the 10 selected candidate effector protein including full length (FL) or without signal peptide (NS) fusion with red fluorescent

protein (RFP). The fluorescence signal was detected with confocal laser-scanning microscope. The result showed that the fluorescence signal of 9 effectors (except CgCFEM3) was clearly detected in N. benthamiana cells (Fig. 4). The FL and NS in most effectors showed similar accumulation in N. benthamiana cells including cell membrane, nucleus, cytosolic bodies and the whole cell. The fluorescence signal of 6 effectors (FL and NS), including CgCFEM1, 4, 7, 9, 14, 15, and 20 were detected in cell membranes. Additionally, CgCFEM1 (FL and NS), CgCFEM4 (NS) and CgCFEM9 (FL) were also accumulated in cytosolic bodies. CgCFEM6 and CgCFEM8 were expressed in nucleus. Moreover, the expression of CgCFEM15 (NS) and CgCFEM20 (NS) were detected in the whole cells including cell membrane, nucleus and cytosolic bodies.

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Ca-Csa2 Mo-ACI1 CFEM1 CFEM2 CFEM3 CFEM4 CFEM5 CFEM6 CFEM7 CFEM8 CFEM9 CFEM10 CFEM11 CFEM12 CFEM13 CFEM14 CFEM15 CFEM16 CFEM17 CFEM18 CFEM19 CFEM20 CFEM21 CFEM22 CFEM23-1 CFEM23-2 CFEM24

Fig. 2 Multiple sequences alignment of amino acid in Common in Fungal Extracellular Membrane (CFEM) domains. CFEM domains of Csa2 (XP_713316.1) from Candida albicans, and ACI1 (AAN64312.1) from Magnaporthe oryzae were used as the positive control. Alignment was performed using ClustalW Program and the result was modified with DNAMAN Software. The conserved amino acid is highlighted in black and gray, and marked with the corresponding letters.

3.4. CgCFEM effectors suppress program cell death in non-host plant Some fungal effector proteins secreted into plant cells to induce or suppress plant cell death, which contribute positively to the virulence of pathogens. Colletotrichum sp., as a typical example, could produce secretary effectors to affect living plant cell, first suppressing cell death in biotrophic stage, then inducing host cell death for necrotrophic stage (Stephenson et al. 2000; Kleemann et al. 2012; Yoshino et al. 2012). To determine whether these CgCFEM effectors have cell death effects on nonhost plant N. benthamiana, we performed transient expression assays via an Agrobacterium-mediated approach. BAX protein from mouse induced programmed cell death (PCD) in N. benthamiana was used as the positive control. Cell death symptoms were observed when N. benthamiana leaves were infiltrated with A. tumefaciens cells carrying PGDR:Bax (Fig. 5). No cell death symptom was observed when individually infiltrated with A. tumefaciens carrying PGDR:FL-CgCFEM or PGDR:NSP-CgCFEM proteins (Fig. 5 and Appendix A). The results indicated that these CgCFEM effectors could not induce cell death in N. benthamiana leaves. On the contrary, co-infiltration of N. benthamiana leaves with A. tumefaciens cells harboring the PGDR:FL-CgCFEM6,

PGDR:FL-CgCFEM7, PGDR:FL-CgCFEM8, PGDR:FLCgCFEM9, PGDR:FL-CgCFEM15 and PGDR:Bax resulted in suppression cell death symptoms at different levels, respectively (Fig. 5). While FL-CgCFEM7, FL-CgCFEM8, FL-CgCFEM9, and FL-CgCFEM15 could completely suppress cell death in the infiltrated area, FL-CgCFEM6 showed partly reduced cell death symptom. Additionally, we further tested the transient expression of these effectors without the presence of signal peptide sequences in N. benthamiana leaves. The results showed that the 5 effectors could still reduce cell death, but the suppression activity of NSP-CgCFEM8 was alleviated compared to full length sequences, suggesting it might be mainly have function in the plant apoplastic space.

4. Discussion CFEM proteins only existed in fungi, and some proteins have been proved as functional effector during the interactions between plants and pathogens (Kulkarni et al. 2003; Zhang et al. 2015). Till now, many CFEM proteins have been identified, and their functions in promoting appressoria formation and pathogenicity have been revealed in some pathogens such as M. oryzae (Kou et al. 2017) and C. albicans (Lamarre et al. 2000; Sabnam and Roy Barman 2017). The composition of aa in CFEM domains were

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Fig. 3 Modeling structural analysis of CgCFEM effectors with Phyre2 server. The amino acid residues of these effectors have been modelled to crystal structure of the CFEM domain in Csa2 from Candida albicans with more than 90% confidence. The amino acid sequence of each effector is indicated in the top line. The sequence is on the next line with residues colored according to property-based scheme: yellow (A, S, T, G, P: small/polar), green (M, I, L, V: hydrophobic), red (K, R, E, N, D, H, Q: charged) and purple (W, Y, F, C: aromatic+cysteine). Green helices represent α-helix, blue arrows indicate β-strands, and faint lines indicate coils. The ‘SS confidence’ line means that red is high confidence and blue is low confidence. The ‘Disorder’ line contains the prediction of disordered regions in the protein by DisoPred, and such regions are indicated by question marks (?). The sequence is scanned against the Conserved Domain Database (CDD) for features of interest. When detected, these are also highlighted as colored dots at the appropriate positions in the sequence with a color key at the bottom of this section (disorder confidence line).

relatively conserved (Kulkarni et al. 2003; Zhang et al. 2015). Each CFEM domain contains a typical 8 characteristically spaced cysteines which could form 4 disulfide bonds to stabilize the protein structure (Zhang et al. 2015; Nasser et al. 2016; Kou et al. 2017). But the CFEM numbers in different fungi were varied. For example, 19 proteins contain 19 CFEM domains in M. oryzae (Kou et al. 2017), 6 proteins contain 9 CFEM domains in C. albicans, 1 protein contains 1 CFEM effector in Saccharomyces cerevisiae and no CFEM domain is detected in Schizosaccharomyces pombe, S. japonicas, Rhizopus oryzae and Batrachochytrium dendrobatidis (Zhang et al. 2015). According to these results, we could clearly find that the CFEM number in pathogenic species was significantly larger than that in non-

pathogenic fungi. There was a positive correlation between CFEM domain occurrence and fungal pathogenicity. The number of CFEM domain identified in C. graminicola was similar to hemibiotrophic pathogen M. oryzae, and both of them are larger than the above-mentioned pathogenicor non-pathogenic fungi. As we know that hemibiotrophic fungi infect plant with biotrophic or necrotrophic stage, and contain more CFEMs than biotrophic or necrotrophic fungi. Hence, we could speculate that these more proteins with special functions may be responsible for the complex pathogenic process of hemibiotrophic fungi. Additionally, we also found that 1 special protein (CgCFEM23) contains 2 CFEM domains in C. graminicola, and they are located in the C-terminal of the amino acid

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CgCFEM14 CgCFEM15 CgCFEM20

Bax

CFEM

Bax+ PGDR

BAX+ CFEM

FE M

7 FE M gC C

C

gC

FE M

6

Fig. 4 Subcellular localization of CgCFEM proteins in Nicotiana benthamiana. The gene of candidate CgCFEM effectors fusion with RFP proteins were cloned into pGDR expression vector and transient expressed in N. benthamiana, respectively. The subcellular localization of each protein (full length and truncated non-signal peptide sequences (No signal P)) was detected with confocal laser-scanning microscope at RFP (with 559 nm excitation and 560 nm emission), bright field, and merged, respectively.

15

CgCFEM9

FE M

CgCFEM8

gC

CgCFEM7

C

CgCFEM6

9

CgCFEM4

FE M

CgCFEM1

gC

Bright Merge

C

No signal P RFP

gC

Bright Merge

C

Full length RFP

8

analyses of CFEM domain, Zhang et al. (2015) found that domain-expansion across fungal genomes appears to be driven by domain duplication and gene duplication via recombination, and CFEMs likely originated in the ancestor of Pezizomycotina, Basidiomycot, and Saccharomycotina and were lost in the ancestor of Taphrinomycotina. These findings generate a clear evolutionary trajectory of CFEM domains and provide novel insights into the functional exchange. These exchanges may create novel biological functions during the whole life stage of fungal strains. Ferric ion is an essential element for plant pathogens especially for pathogen virulence. Colletotrichum graminicola infection progressed rapidly via biotrophic to necrotrophic growth in Fe-deficient leaves, while an adequate Fe nutritional status suppressed the formation of infection structures of fungi during the early biotrophic growth phase. The regulation of Fe nutrition in suitable condition is important for plant pathogens (Ye et al. 2014). Till now, several iron uptake systems of fungi have been identified such as reductive iron assimilation, siderophore-mediated Fe3+ uptake, heme uptake and direct Fe2+ uptake (Albarouki and Deising 2013). It has been proved that CFEM domain containing proteins contain iron acquisition activity in C. albicans. Exactly, the CFEM domain could form a helicalbasket fold structure, and 1 aspartic residue (D80) serves as the CFEM axial ligand which could coordinate with Fe3+ haem. If it was replaced by His residue, the protein could acquire Fe2+ haem instead of Fe3+ haem (Nasser et al. 2016). Except for an aspartic acid side chain axial ligand, some other amino acids including His, Met, Cys, Tyr and Lys have also been observed for the coordination of iron. In C. graminicola, the aspartic residue (D80) was relatively conserved in 12 CFEM domains (Fig. 2; CFEM1, 3, 4, 6, 9, 12, 13, 14, 15, 22, 23-2 and 24), in other 9 domains (Fig. 2;

sequences. This phenomenon was also found in few other fungi such as Arthrobotrys oligospora, Botryotinia fuckeliana. What is the reason for the different composition or number of CFEMs in fungi? According to the systematic

FL

NSP

Fig. 5 Transient expression analysis of 5 CgCFEM in Nicotiana benthamiana leaves with suppressing cell death activity by using an agroinfiltration approach. Agroinfiltration was performed on tobacco leaf with Agrobacterium tumefaciens carrying PGDR:Bax gene (BAX), PGDR:CgCFEM genes (CFEM), BAX, as the positive control, was infiltrated with candidate CFEM to detect the hypersensitive response, respectively. The results were recorded 5 days post inoculation (dpi), respectively. FL, full length of CgCFEM sequences in transient expression; NSP, truncated non-signal peptide sequences.

GONG An-dong et al. Journal of Integrative Agriculture 2020, 19(2): 541–550

CFEM2, 5, 10, 11, 16, 17, 18, 19 and 20), it was replaced by asparagine residue (N80). Four domains were replaced with Thr (Fig. 2; CFEM7), Ile (Fig. 2; CFEM8), Phe (Fig. 2; CFEM21), Gly (Fig. 2; CFEM23-1), respectively. The variation of this conserved amino acid among these CFEM domains may provide variable iron acquisition in the life cycle of C. graminicola. This replication might cause different iron acquisition activity of C. graminicola in plant, which cause different pathogenicity of fungi in plant. The selected 10 candidate effectors may have predominant functions during the pathogenicity located in cell membrane, cell nucleus, and the whole cells. Additionally, the replacement of conserved amino acid in CFEM domain may cause different iron affinity, which plays important roles in the pathogenic process. In our further study, we would analyze the functions of these candidate CgCFEM proteins, and identified their iron-binding activity associated with pathogenicity.

5. Conclusion Here, we identified 24 CgCFEM proteins containing 25 CFEM domains in the genome of C. graminicola. Ten of the 24 CgCFEM proteins were considered as candidate effectors due to the presence of signal peptide and lack of trans-membrane domains. These effectors could locate in cell membrane, nucleus and the whole cells, and 5 of them could suppress PCD on tobacco leaves. Moreover, the CFEM domain in these effectors could form a helical-basket structure with different conserved amino acid, which might be responsible for iron acquisition and pathogenicity. These candidate effectors with varied localization, structure, and biological activity may endow important functions during the pathogenic processes of hemibiotrophic fungi.

Acknowledgements We thank Dr. Martin Dickman in Texas A&M University for providing the C. graminicola strain CgM2. This study was supported by the National Program for Support of Top-notch Young Professionals of China. Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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