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Fusarium blight of wheat is usually caused by Fusarium graminearum, and the pathogenic fungi will secrete effectors into the host plant tissue to affect its normal physiological process, so as to make it pathogenic. The CFEM (Common in Fungal Extracellular Membrane) protein domain is unique to fungi, but it is not found in all fungi. The CFEM protein contained in F. graminearum may be closely related to pathogenicity. In this study, 23 FgCFEM proteins were identified from the F. graminearum genome. Then, features of these proteins, such as signal peptide, subcellular localization, and transmembrane domains, etc., were analyzed and candidate effectors were screened out. Sequence alignment results revealed that each FgCFEM protein contains one CFEM domain. The amino acids of the CFEM domain are highly conserved and contain eight spaced cysteines, with the exception that FgCFEM8, 9, and 15 lack two cysteines and three cysteines were missed in FgCFEM18 and FgCFEM22. A recently identified CFEM_DR motif was detected in 11 FgCFEMs, and importantly we identified two new conserved motifs containing about 29 and 18 amino acids (CFEM_WR and CFEM_KF), respectively, in some of FgCFEM proteins. Transcriptome analysis of the genes encoding CFEM proteins indicated that all the CFEM-containing genes were expressed during wheat infection, with seven and six genes significantly up- and down-regulated, respectively, compared with in planta and in vitro. Based on the above analysis, FgCFEM11 and FgCFEM23 were predicted to be F. graminearum effectors. This study provides the basis for future functional analyses of CFEM proteins in F. graminearum.

SummaryMechanims required for broad spectrum or specific host colonization of plant parasites are poorly understood. As a perfect illustration, heteroecious rust fungi require two alternate host plants to complete their life cycle. Melampsora larici-populina infects two taxonomically unrelated plants, larch on which sexual reproduction is achieved and poplar on which clonal multiplication occurs leading to severe epidemics in plantations. High-depth RNA sequencing was applied to three key developmental stages of M. larici-populina infection on larch: basidia, pycnia and aecia. Comparative transcriptomics of infection on poplar and larch hosts was performed using available expression data. Secreted protein was the only significantly over-represented category among differentially expressed M. larici-populina genes in basidia, pycnia and aecia compared together, highlighting their probable involvement in the infection process. Comparison of fungal transcriptomes in larch and poplar revealed a majority of rust genes commonly expressed on the two hosts and a fraction exhibiting a host-specific expression. More particularly, gene families encoding small secreted proteins presented striking expression profiles that highlight probable candidate effectors specialized on each host. Our results bring valuable new information about the biological cycle of rust fungi and identify genes that may contribute to host specificity.

Fusarium blight of wheat is usually caused by Fusarium graminearum, and the pathogenic fungi will secrete effectors into the host plant tissue to affect its normal physiological process, so as to make it pathogenic. The CFEM (Common in Fungal Extracellular Membrane) protein domain is unique to fungi, but it is not found in all fungi. The CFEM protein contained in F. graminearum may be closely related to pathogenicity. In this study, 23 FgCFEM proteins were identified from the F. graminearum genome. Then, features of these proteins, such as signal peptide, subcellular localization, and transmembrane domains, etc., were analyzed and candidate effectors were screened out. Sequence alignment results revealed that each FgCFEM protein contains one CFEM domain. The amino acids of the CFEM domain are highly conserved and contain eight spaced cysteines, with the exception that FgCFEM8, 9, and 15 lack two cysteines and three cysteines were missed in FgCFEM18 and FgCFEM22. A recently identified CFEM_DR motif was detected in 11 FgCFEMs, and importantly we identified two new conserved motifs containing about 29 and 18 amino acids (CFEM_WR and CFEM_KF), respectively, in some of FgCFEM proteins. Transcriptome analysis of the genes encoding CFEM proteins indicated that all the CFEM-containing genes were expressed during wheat infection, with seven and six genes significantly up- and down-regulated, respectively, compared with in planta and in vitro. Based on the above analysis, FgCFEM11 and FgCFEM23 were predicted to be F. graminearum effectors. This study provides the basis for future functional analyses of CFEM proteins in F. graminearum.

Background Gymnosporangium spp. are fungal plant pathogens causing rust disease and most of them are known to infect two different host plants (heteroecious) with four spore stages (demicyclic). In the present study, we sequenced the transcriptome of G. japonicum teliospores on its host plant Juniperus chinensis and we performed comparison to the transcriptomes of G. yamadae and G. asiaticum at the same life stage, that happens in the same host but on different organs. Results Functional annotation for the three Gymnosporangium species showed the expression of a conserved genetic program with the top abundant cellular categories corresponding to energy, translation and signal transduction processes, indicating that this life stage is particularly active. Moreover, the survey of predicted secretomes in the three Gymnosporangium transcriptomes revealed shared and specific genes encoding carbohydrate active enzymes and secreted proteins of unknown function that could represent candidate pathogenesis effectors. A transcript encoding a hemicellulase of the glycoside hydrolase 26 family, previously identified in other rust fungi, was particularly highly expressed suggesting a general role in rust fungi. The comparison between the transcriptomes of the three Gymnosporangium spp. and selected Pucciniales species in different taxonomical families allowed to identify lineage-specific protein families that may relate to the biology of teliospores in rust fungi. Among clustered gene families, 205, 200 and 152 proteins were specifically identified in G. japonicum, G. yamadae and G. asiaticum, respectively, including candidate effectors expressed in teliospores. Conclusions This comprehensive comparative transcriptomics study of three Gymnosporangium spp. identified gene functions and metabolic pathways particularly expressed in teliospores, a stage of the life cycle that is mostly overlooked in rust fungi. Secreted protein encoding transcripts expressed in teliospores may reveal new candidate effectors related to pathogenesis. Although this spore stage is not involved in host plant infection but in the production of basidiospores infecting plants in the Amygdaloideae, we speculate that candidate effectors may be expressed as early as the teliospore stage for preparing further infection by basidiospores.

Pathogens secrete effector proteins to suppress host immunity, mediate nutrient uptake and subsequently enable parasitism. However, on non-adapted hosts, effectors can be detected as non-self by host immune receptors and activate non-host immunity. Nevertheless, the molecular mechanisms of effector triggered non-host resistance remain unknown. Here, we report that a small cysteine-rich protein PstSCR1 from the wheat rust pathogen Puccinia striiformis f. sp. tritici (Pst) activates immunity in the non-host solanaceous model plant Nicotiana benthamiana. PstSCR1 homologs were found to be conserved in Pst, and in its closest relatives, Puccinia graminis f. sp. tritici and Puccinia triticina. When PstSCR1 was expressed in N. benthamiana with its signal peptide, it provoked the plant immune system, whereas no stimulation was observed when it was expressed without its signal peptide. PstSCR1 expression in N. benthamiana significantly reduced infection capacity of the oomycete pathogens. Moreover, apoplast-targeted PstSCR1 triggered plant cell death in a dose dependent manner. However, in Brassinosteroid insensitive 1-Associated Kinase 1 (SERK3/BAK1) silenced N. benthamiana, cell death was remarkably decreased. Finally, purified PstSCR1 protein activated defence related gene expression in N. benthamiana. Our results show that a Pst-secreted protein, PstSCR1 can activate surface mediated immunity in non-adapted hosts and contribute to non-host resistance.

Uromyces fabae is a major pathogen of broad bean, Vicia faba. U. fabae has served as a model among rust fungi to elucidate the development of infection structures, expression and secretion of cell wall degrading enzymes and gene expression. Using U. fabae, enormous progress was made regarding nutrient uptake and metabolism and in the search for secreted proteins and effectors. Here, we present results from a genome survey of U. fabae. Paired end Illumina sequencing provided 53 Gb of data. An assembly gave 59,735 scaffolds with a total length of 216 Mb. K-mer analysis estimated the genome size to be 329 Mb. Of a representative set of 23,153 predicted proteins we could annotate 10,209, and predict 599 secreted proteins. Clustering of the protein set indicates families of highly likely effectors. We also found new homologs of RTP1p, a prototype rust effector. The U. fabae genome will be an important resource for comparative analyses with U. appendiculatus and P. pachyrhizi and provide information regarding the phylogenetic relationship of the genus Uromyces with respect to other rust fungi already sequenced, namely Puccinia graminis f. sp. tritici, P. striiformis f. sp. tritici, Melampsora lini, and Melampsora larici-populina.

Melampsora larici-populina is a fungal pathogen responsible for foliar rust disease on poplar trees, which causes damage to forest plantations worldwide, particularly in Northern Europe. The reference genome of the isolate 98AG31 was previously sequenced using a whole genome shotgun strategy, revealing a large genome of 101 megabases containing 16,399 predicted genes, which included secreted protein genes representing poplar rust candidate effectors. In the present study, the genomes of 15 isolates collected over the past 20 years throughout the French territory, representing distinct virulence profiles, were characterized by massively parallel sequencing to assess genetic variation in the poplar rust fungus. Comparison to the reference genome revealed striking structural variations. Analysis of coverage and sequencing depth identified large missing regions between isolates related to the mating type loci. More than 611,824 single-nucleotide polymorphism (SNP) positions were uncovered overall, indicating a remarkable level of polymorphism. Based on the accumulation of non-synonymous substitutions in coding sequences and the relative frequencies of synonymous and non-synonymous polymorphisms (i.e., PN/PS ), we identify candidate genes that may be involved in fungal pathogenesis. Correlation between non-synonymous SNPs in genes encoding secreted proteins (SPs) and pathotypes of the studied isolates revealed candidate genes potentially related to virulences 1, 6, and 8 of the poplar rust fungus.

Background Plant and animal pathogenic fungus Cochliobolus lunatus cause great economic damages worldwide every year. C. lunatus displays an increased temperature dependent-virulence to a wide range of hosts. Nonetheless, this phenomenon is poorly understood due to lack of insights on the coordinated secretome weaponries produced by C. lunatus under heat-stress conditions on putative hosts. To understand the mechanism better, we dissected the secretome of C. lunatus interacting with potato (Solanum tuberosum L.) leaf at different temperature regimes. Results C. lunatus produced melanized colonizing hyphae in and on potato leaf, finely modulated the ambient pH as a function of temperature and secreted diverse set of proteins. Using two dimensional gel electrophoresis (2-D) and mass spectrometry (MS) technology, we observed discrete secretomes at 20°C, 28°C and 38°C. A total of 21 differentially expressed peptide spots and 10 unique peptide spots (that did not align on the gels) matched with 28 unique protein models predicted from C. lunatus m118 v.2 genome peptides. Furthermore, C. lunatus secreted peptides via classical and non-classical pathways related to virulence, proteolysis, nucleic acid metabolism, carbohydrate metabolism, heat stress, signal trafficking and some with unidentified catalytic domains. Conclusions We have identified a set of 5 soluble candidate effectors of unknown function from C. lunatus secretome weaponries against potato crop at different temperature regimes. Our findings demonstrate that C. lunatus has a repertoire of signature secretome which mediates thermo-pathogenicity and share a leucine rich “CL[xxxx]LHM”-motif. Considering the rapidly evolving temperature dependent-virulence and host diversity of C. lunatus, this data will be useful for designing new protection strategies.