Functional Analyse Of SshPpi Gene In Scleromitula Shiraiana

Mulberry is the fruit of mulberry tree,which contains many essential nutrients for human body.It has good edible and medicinal value,and has great prospects of development and utilization.Mulberry sclerotial disease is the most harmful fungal disease in the mulberry production,the incidence rate of serious areas can reach 90%,which has a serious impact on the mulberry industry.Mulberry shrunken fruit disease is caused by the infection of Scleromitula shiraiana,which is one of the most important fungal diseases affecting mulberry production.The infected mulberries are shriveled and pale in color.Meanwhile,the mycelium intertwines to form a hard black sclerotium.In the second year,the germination of sclerotium forms a secondary infection,which brought great harm to the mulberry production.At present,the prevention and control of mulberry sclerotial disease is mainly based on prevention and there is no good coping strategy,which has caused great obstacles to the development of mulberry industry.Effectors are small secreted proteins that play key roles in pathogenicity,growth and development,metabolite synthesis,and abiotic stress of pathogenic fungi.The study of effectors can further elucidate the mechanism of pathogen-plant interaction.At present,many effectors of pathogenic fungi have been analysed,but the research in S.shiraiana is relatively weak due to its late start.Sclerotinia sclerotiorum is a kind of necrotrophic fungi with wide host range and great harm.S.sclerotiorum and S.shiraiana belong to the family sclerotiniaceae and are closely related.In addition,the genetic transformation system of S.sclerotiorum is mature.And the pathogenic mechanism of S.sclerotiorum is studied further.So it is possible to better study the S.shiraiana by associating them.In this thesis,an effector SshPpi of S.shiraiana was studied in depth,which provided basic data for clarifying the molecular mechanism of effector of S.shiraiana and realizing the green prevention and control of mulberry sclerotial disease.The main research results are as follows:1.Screening the effectorsFifteen potential effectors were screened using Agrobacterium-mediated transient expression system.An effector SshPpi(promote pathogen infection)was obtained by screening,which could significantly promote the infection of S.sclerotiorum on Nicotiana benthamiana leaves.It was speculated that this protein was closely related to the pathogenic process of the pathogen.2.Basic characteristic studySshPpi encoded 218 amino acids and contains a signal peptide of 21 amino acids at the N-terminal.There is no transmembrane domain,conserved structural domain and GPI anchor point.Its homologous proteins are widely found in pathogenic fungi.The homologous protein SscPpi of S.sclerotiorum is closely related to SshPpi and highly similar in structure.3.Functional verification of the SshPpi gene of S.shiraianaThe overexpression of SshPpi in S.sclerotiorum by PEG mediated genetic transformation was carried out.The phenotypes of the overexpression strains were further analyzed.In terms of growth and development,there were no significant differences in mycelium growth rate,colony morphology,sclerotinia formation,and sclerotinia dry weight between SshPpi overexpression strains and wild-type S.sclerotiorum.In terms of pathogenicity,SshPpi overexpression strains could produce oxalic acid normally and induce the formation of infection pads.Compared with wild-type S.sclerotiorum,Sshppi overexpression strains formed more infection pads.It’s pathogenicity to N.benthamiana in vitro was significantly enhanced.In terms of abiotic stress,SshPpi overexpressing strains did not respond to oxidative stress.Compared with the wild-type strains,SshPpi overexpressing strains was more sensitive to hypertonic stress(KCl,Na Cl and sucrose).The results of yeast two-hybrid showed that SshPpi did not interact with 16 proteins(Ma PR1 、 Ma PR2 、 Ma PR3 、 Ma PR5 、Ma NPR1 、 Ma SAR1 、 Ma EDS5 、 Ma PAD4 、 Ma SNI1 、 Ma ICS1 、 Ma PAL2 、Ma PR5CBP60g、Ma CUL3、Ma NPR1-like2、Ma PR1-like3 and Ma TGA3)related to SA signaling pathway.These results indicated that SshPpi did not participate in the vegetative growth of S.sclerotiorum,but was involved in the pathogenic process and hypertonic stress of S.sclerotiorum.4.Functional verification of the SscPpi gene of S.sclerotiorumThe homologous gene SscPpi of SshPpi was knocked out by PEG mediated genetic transformation,and the phenotypes of knockout strains were analyzed in detail.In terms of growth and development,SscPpi gene knockout strains showed no significant differences in colony morphology,sclerotium formation and sclerotium dry weight compared with wild-type strain,but the mycelium growth rate was slower.In terms of pathogenicity,SscPpi knockout strains could normally produce oxalic acid and induce the formation of infection pads.However,the infection pads formed by the SscPpi knockout strains were less than those of the wild-type strain and the pathogenicity to N.benthamiana in vitro was also significantly reduced.In abiotic stress,SscPpi knockout strains did not respond to oxidative stress and some hypertonic stresses(Na Cl and sucrose).Compared with the wild-type strain,it was more tolerant to the hypertonic environment formed by KCl.These results indicated that SscPpi was involved in mycelium development,but not in sclerotinia development.Sscppi was involved in the pathogenesis and hypertonic stress of sclerotinia.These results indicate that the SshPpi protein of S.shiraiana is a new effector,which is widely distributed in pathogenic fungi and participates in the pathogenicity of pathogens.The results of this study provide a theoretical basis for clarifying the role of Ppi effectors in the pathogenic process and the green prevention and control of mulberry shrunken fruit disease.