Comparative Genomic,Transcriptomic And Gene Function Studies Reveal The Molecular Mechanisms Of Pathogenesis And Development Of Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.) de Bary is a typical necrotrophic phytopathogenic fungus with a broad host range, which could attack at least 408 species of plants and subspecies. S. sclerotiorum is also a cosmopolitan pathogen of many economically important crops, including rapeseed, sunflowers, soybeans, lentils, lettuces and carrots and its infection often leads to a significant loss of crop production.In order to study the development of S. sclerotiorum on genome level, the genome of S. sclerotiorum was functionally annotated in silico and comparative genomic analysis was performed between S. sclerotiorum and the other fungi sequenced. Our results showed that the numbers of the CENP-B-like DDE superfamily endonucleases and reverse transcriptases associated with transposable elements(TEs) in S. sclerotiorum are much larger than that in other species, indicating TEs expansion has strongly influenced the evolutionary trajectory and genome organization of S. sclerotiorum. The categories of related TEs and their relative positions with CENP-B-like DDE superfamily endonuclease genes and reverse transcriptase genes in S. sclerotiorum genome were further analyzed to show that these two gene family were indeed related to TEs, and most of CENP-B-like DDE superfamily endonuclease genes are related to the repeat class/family of DNA/TcMar-Fot1; All reverse transcriptase genes are related to RNA TEs, most of which are the repeat classes/families of LINE/Tad1, LTR/Gypsy and LTR/Copia. Through comparison of the types and numbers of TEs in S. sclerotiorum and other fungi, we found both of DNA TEs and RNA TEs played important roles in the evolution of S. sclerotiorum genome. Phylogenetic analyses revealed the expansion of CENP-B-like DDE superfamily endonuclease family and reverse transcriptase family may be caused by the dramatical duplication of corresponding TEs in a short time in the evolution of S. sclerotiorum genome. Transcriptional analysis showed that the majority of these two kinds of TEs had been "inactivated", while only a few of them are still “active”. Insertion into the downstream of host genes may be one way of maintaining their “activity”.The digital gene expression(DGE) system was initially used to gain an insight into the wide range of transcriptional responses associated with six key developmental stages of S. sclerotiorum including vegetative growth, infection, sclerotial development, myceliogenic germination of sclerotia, carpogenic germination of sclerotia and apothecium formation. To globally understand the dynamic change of various gene functional modules on different levels during S. sclerotiorum development, a method namely “functional profile analysis” was developed according to the measure of relative expression levels of various functional modules such as gene ontology(GO) and Interpro categories as well as KEGG pathways. “Functional profile analysis” combined the gene expression level with the gene functional annotation to illustrate the activity of corresponding functional modules. Through the comparison of “functional profiles”, we could detect the dynamic change of the relative activity of various functional modules on different levels during different developmental stages of S. sclerotiorum.Our functional profile analysis showed an incomplete CFPP-like pathway(CLP) was significantly activated during the infection, sclerotial myceliogenic germination and sclerotial carpogenic germination of S. sclerotiorum, and this CLP was selected for further study. Carbon fixation pathway of plants(CFPP) in photosynthesis converts solar energy to biomass, bioproducts and biofuel. Intriguingly, a large number of heterotrophic fungi also possess enzymes functionally associated with CFPP, raising the questions about their roles in fungal development and in evolution. We found the presence of 17 CFPP associated enzymes [ten in Calvin-Benson-Basham(CBB) reductive pentose phosphate pathway and seven in C4-dicarboxylic acid cycle] in the genome of S. sclerotiorum, a heterotrophic phytopathogenic fungus, and only two unique enzymes: ribulose-1, 5-bisphosphate carboxylase-oxygenase(Rubisco) and phosphoribulokinase(PRK) were absent in CBB cycle. This data suggested an incomplete CFPP-like pathway(CLP) in fungi. Functional profile analysis demonstrated that the activity of the incomplete CLP was dramatically regulated during different developmental stages of S. sclerotiorum. Subsequent experiments confirmed that many CLP associated enzymes were essential to the virulence and/or sclerotial formation. Most of the CLP associated genes are conserved in fungi. Phylogenetic analysis showed that many of them have undergone gene duplication, gene acquisition or loss and functional diversification in evolutionary history. These findings showed an evolutionary link in the carbon fixation processes of autotrophs and heterotrophs and implicated the functions of related genes were in course of continuous change in different organisms in evolution.The functional modules associated with carbohydrate-active enzymes(CAZymes) were also significantly induced during the sclerotial development and infection of S. sclerotiorum. Our comparative genomic analysis showed that the numbers of plant cell wall(PCW)- and fungal cell wall(FCW)-degradation-associated carbohydrate-active enzymes(CAZymes) in necrotrophic and hemibiotrophic fungi are significantly larger than that in most biotrophic fungi. However, our transcriptional analyses of CAZyme-encoding genes in S. sclerotiorum, Fusarium graminearum, Melampsora larici-populina and Puccinia graminis showed that many genes encoding PCW- and FCW-degradation associated CAZymes were significantly up-regulated during infection of both necrotrophic fungi and biotrophic fungi, indicating an existence of a universal mechanism underlying PCW degradation and FCW reorganization or modification, which are both intimately involved in necrotrophic and biotrophic fungal infection. Furthermore, our results showed that the FCW reorganization or modification was also related to the fungal development.Our transcriptomic analysis showed many secreted protein-encoding genes were significantly up-regulated during the sclerotial development and infection of S. sclerotiorum. Small, secreted proteins have been found to play crucial roles in interactions between biotrophic/hemi-biotrophic pathogens and plants. However, little is known about the roles of these proteins produced by necrotrophic plant pathogens during infection. As examples, two cysteine-rich, small, secreted protein(SsCVNH and SsSSVP1) were experimentally confirmed to be essential for the full virulence of S. sclerotiorum. Further study showed that the secretion of SsSSVP1 from hyphae was followed by internalization and cell-to-cell movement independent of a pathogen in host cells. SsSSVP1 primarily localized in cytoplasm of the host cells where it induced significant host cell death. SsSSVP1 was dramatically up-regulated during infection and targeted silencing of SsSSVP1 resulted in a significant reduction in virulence. Through yeast two-hybrid(Y2H), coimmunoprecipitation(co-IP) and bimolecular fluorescence complementation(Bi FC) assays, we demonstrated that SsSSVP1 interacted with QCR8, a subunit of the cytochrome b-c1 complex of mitochondrial respiratory chain in plants. Double site-directed mutagenesis of two cysteine residues(C38 and C44) had a significant effect on the dimer formation of SsSSVP1, Ss SSVP1-QCR8 interaction and plant cell death induced by SsSSVP1, indicating that partial cysteine residues play crucial roles in maintaining the structure and function of SsSSVP1. Co-localization and BiFC assays showed that SsSSVP1 might hijack QCR8 to cytoplasm thereby disturbing its subcellular localization before QCR8 targeting into mitochondria. Furthermore, virus induced gene silencing(VIGS) of QCR8 in Nicotiana benthamiana caused plant abnormal development and cell death, indicating the cell death induced by SsSSVP1 might be caused by the SsSSVP1-QCR8 interaction, which had disturbed the subcellular localization of QCR8 and hence interfered with the biological functions of QCR8 in mitochondria. These results indicated that the small secreted proteins might also play crucial roles as potential effectors in host-non-specific necrotrophic fungi. Our findings reveal novel roles of the small secreted proteins in the interactions between broad host range necrotrophic fungi and plants, and highlight the significance to illuminate the pathogenic mechanisms of this type of interactions.In this study, the molecular mechanisms of pathogenesis and development of S. sclerotiorum were explored on different levels including genome, transcriptome and single gene. The results indicated that all developmental stages of S. sclerotiorum are affected by networks consisting of multiple genes which have functional links and play roles synergistically, however, the exact function and status of each gene in these networks are different. Some key genes play decisive roles independently in certain biological process. In this study, we combined the DGEs of S. sclerotiorum with bioinformatics analyses to explore the molecular mechanisms of various biological processes of S. sclerotiorum from two aspects: on one hand, the overall performance of various functional modules was analyzed in a macroscopic view; on the other hand, the biological functions of some key genes were in-depth studied in a microscopic view. Through these two strategies, this study demonstrated a comprehensive understanding on the molecular mechanisms of the pathogenesis and development of S. sclerotiorum and provided new perspectives and theoretical supports for the disease control of S. sclerotiorum.