Molecular Mechanisms Underlying Effect Of Mycelial Age On Pathogenicity Of Sclerotinia Sclerotiorum And Plant Resistance To This Fungus

Sclerotinia sclerotiorum is one of the world wide distributed necrotrophic fungal pathogens. The white mould disease caused by this fungus is an important disease of oil and vegetable crops. Oxalic acid (OA) and cell wall degrading enzymes (CWDEs) are the key pathogenecity factors of this fungus. When meets limited space or nutrition conditons, aerial hyphae will generate and finally sclerotia formed. This morphological change leads to alteration of pathogenecity. However, the mechanism of this change is still unclear. Currently, the mechanisms underlying the plant resistance to S.sclerotiorum remain largely unknown. Thus identification of genes involved in this resistance is urgently needed and the nonhost resistance of rice to S. scelerotiorum may provide a new insight to plant resistance to this pathogen. In this study, we elucidated the mechanisms of interaction between plant and S.sclrotiorum by dissecting the mechanism of effect of mycelia age on fungal pathogenecity, revealing the role of plant Ca2+signaling pathways in resistance to S.sclerotiorum, employing Nicotiana benthamiana cDNA library based VIGS screening to obtain novel regulatory genes of S. sclerotiorum resistance and revealing the interaction between rice and S. sclerotiorum..The main results are as follows.(1) The influence of morphology and age of hyphae on pathogenicity of S.sclerotiorum and the underlying mechanism was revealed. Plenty of aerial hyphae formed from mature hyphae when grown in medium plates. Aerial hyphae were much smaller in diameter, only1/5of that of young vegetative hyphae. When compared with young hyphae, the virulence of1d-3d-old mycelia dramatically decreased. Further analysis showed that expression of OAH, which encodes oxaloacetate acetylhydrolase, the key enzyme to catalyze oxalic acid (OA) biosynthesis, in old mycelia was strongly repressed, and the ability to secrete OA was also reduced dramatically in the old mycelia. Exogeneously supply with OA (10mM, pH7.0) enabled the old mycelia infect the host plants. These results demonstrate that loss of virulence of old mycelia is mainly caused by being unable to accumulate OA. Moreover, superoxide anion and hydrogen peroxide (H2O2) accumulated less in1d-3d-old hyphae than0d-old hyphae, which was coincided with the expression pattern of ROS metabolism related genes in hyphae at different ages. Additionally, RNAi analysis showed that silencing of EB1gene, which encodes an EBl-like microtubule-associated protein, retarded the fungal growth and development and reduced sclerotium formation as well as virulence in plants, indicating that EB1plays an important role in the fungal growth and development, sclerotium formation and virulence in plants. (2) The role of calcium signaling pathway and AtWRKY11in resistance to S.sclerotiorum was elucidated. Expression profiles were revealed for rapeseed leaves inoculated with S.sclerotiorum, which showed that expression of1072genes altered by over2folds, among which531genes were up regulated while541genes were down regulated after inoculation. Interestingly,15differentially expressed genes were related with calcium signaling pathways. Pharmarcological study showed that infiltration with Ca2+channel blocker LaCl3(1mmol/L), Ca2+ATPase blocker NaVO3(50μmol/L) reduced the resistance to S. sclerotiorum in Nicotiana benthamiana, while treatment with CaCl2(10mmol/L) enhanced this resistance. Treatment with these regulators significantly induced the expression of a set of calcium signaling genes including AtWRKYll. The Arabidopsis wrkyll mutant exhibited obviously decreased resistance to S. sclerotiorum. These results reveal that calcium signaling pathway is involved in plant resistance to S. sclerotiorum.(3) The Calcium-dependent protein kinase (CDPK) and CDPK-related protein kinase (CRK) gene family was identified in tomato at genome-wide level, and the function of SICDPK and SICRK in resistance to S. sclerotiorum was revealed employing virus induced gene silencing (VIGS) technique. Twenty nine CDPK genes and six CRK genes were identified in tomato genome employing bioinformatics approaches. All S1CDPK proteins harbor a STKc CAMK type Serine/Threonine protein kinase domain and two EF-hand type Ca2+binding domains, while SICRK proteins solely contain a STKc CAMK type protein kinase domain at their N termini and degenerated EF-hand like sequence at their C termini. Phylogenetic analysis revealed that the tomato CDPK family was classified into four subgroups. Among them, subgroup IV S1CDPKs were unique that their proteins were basic and their genes contained11introns. Furthermore, functional analyses employing virus-induced gene silencing showed that SICRK6was involved in the resistance to both S. sclerotiorum and Pst DC3000in tomato, while SICDPK10and SICDPK18did not affect the resistance to S. sclerotiorum, rather positively regulated the resistance to Pst DC3000and Xoo respectively. Our results reveal that SICDPK and SICRK genes regulate a wide range of resistance in tomato but the effective pathogens are gene-dependent. Moreover, yeast two hybrid analysis showed that SICDPK12interacted with SlCaML.(4) A number of genes involved in regulating the OA tolerance and S. sclerotiorum resistance were obtained employing N. benthamiana cDNA library based VIGS screening.511colonies expressing VIGS constructs were ramdomly selected for VIGS functional analyses. Finally,71genes involved in OA tolerance and63genes participating in S. sclerotiorum resistance were screened out. Silencing of genes including Upstream activation factor subunit UAF30(No.437) and Translationally-controlled tumor protein (No.502) compromised the OA tolerance as well as S. sclerotiorum resistance, while silencing of genes such as Aspartic proteinase oryzasin-1(No.135) enhanced these abilities, indicating that these genes may play an important role in OA-mediated plant-S. sclerotiorum interactions. However, some genes were only involved in OA tolerance or S. sclerotiorum resistance, implying that there are OA-independent mechanisms of resistance to S. sclerotiorum.(5) The rice-S. sclerotiorum nonhost resistance system was established, and its underlying mechanism was probed. HR-like small brown necrotic spots appeared in rice leaves at24h post inoculation with S. sclerotiorum wild type strain1980. High level of ROS accumulated inside the brown spots, and the fungal mycelia were confined to the HR area. These symptoms did not appear in leaves inoculated with the non-pathogenic PB strain. Quantitative proteomics analysis was performed employing TMT labeling technique. Comparative analysis for differentially expressed proteins between leaves inoculated with S. sclerotiorum strain1980and PB as well as between those inoculated with strain PB and agar plugs revealed a set of proteins involved in rice resistance to S. sclerotiorum. These included P-protein; Proline-rich-like protein; ribonuclease T2family protein; RALFL4(Rapid ALkalinization Factor RALF family protein); lrgB-like family protein and CHIT14(Chitinase family protein) as well we a set proteins related with oxareduction. Among these, RALFL4has a function in alkalinization and CHIT14may degrade chitin of fungal cell walls. These data support that maintenance of acidic status is required for nonhost resistance to S. sclerotiorum in rice. In addition, the differentially expressed Eukaryotic translation initiation factor3was identified as a ubiquitinated protein, indicating that protein ubiquitylation is involved in resistance to S. sclerotiorum.