Study On Function And Mechanism Of Two Kinds Of Valsa Mali Effectors VmHEPs And VmPODs

The apple industry is a major agro-industry in China.It is seriously threatened by the apple Valsa canker which is a devastating disease caused by the Valsa mali.However,the mechanism of infection and interaction are still unkown and need to be revealed urgently.Effector proteins,acting as an important pathogenic factor of plant pathogenic fungi,play essential roles in the interaction with host plants.The effectors identified from fungi are differet from those in oomycetes and bacteria.Most of the fungal effectors have no conserved domains.However,the conserved domains always indicate potential feature of functions which are essential.Hce2 domain is one of the most widely distributed and important effector conserved domains identified in plant pathogenic fungi,but its mechanism and mode of action are still unknown;Pod?Peroxidase?doamain is well study in its metabolism and physiological function in organism,but if it could act as effectors during the interation between plant pathogenic fungi and host is still unrevealed.Especially,we still know nothing about effectors containing conserved domain in V.mali.This research identified and studied on function of two kinds of effectors containing conserved domains,the VmHEPs?VmHEP1,VmHEP2,VmHEP3,VmHEP4 and VmHEP5?which contain Hce2 domain and the VmPODs?VmPOD1,VmPOD2 and VmPOD3?which contains Pod domain.The main results are summarized as follows:?1?Hce2 domain-containing effectors VmHEPs contributed to full virulence of V.mali in a gene redundant mannerIn this study,we identified one cell death inducer VmHEP1,containing Hce2 domian,from 88 candidate secreted proteins and four homologous candidate effectors?VmHEP2,VmHEP3,VmHEP4 and VmHEP5?were identified as the homologous protein of VmHEP1.Those five genes are paralogous genes and under purify selection,VmHEP1 and VmHEP2are symparalogs?in-paralogs?,while the other three genes are alloparalogs?out-paralogs?and only VmHEP1 could lead to cell death on the leaves of Nicotiana benthamiana.Deletion of each single gene of VmHEPs did not affect virulence of V.mali.Intringuing,double deletion of both VmHEP1 and VmHEP2 led to significantly virulence reduction on both leaves and twigs.Further analysis showed VmHEP1 and VmHEP2 were tandem gene and closed to each other with on 604 bp distance on chromosome 11.What is more,the qRT-PCR showed the deletion of VmHEP1 will induce the up-regulated expressing of VmHEP2 and the deletion of VmHEP2 could also stimulate the expression of VmHEP1.The results revealed effectors containing Hce2 domian from V.mali,acting as an important virulence factors in a redundant manner and provided insights into the role of tandem effectors in virulence during the interaction between pathogenic fungi and plant.?2?The upstream non-coding region of VmHEP1 gene has a promoter function induced by the infection and effector VmHEP1 can interact with the apple protein MdLRRP1We further researched the upstream non-coding region function and identified the interaction target of VmHEP1 which was the only cell death-inducer in this study.The genome analysis showed unpstream 1500 bp of VmHEP1 was non-coding region.Basing on the genetic transformation system of V.mali,we confirmed that the 1500 bp upstream non-coding region can active the expression of exogenous GFP gene which indicated potential promoter function and the followed qRT-PCR showed the potential promoter was induced during infection.Further,by means of yeast two hybrid?Y2H?,co-immunoprecipitation assay?Co-IP?and mass spectrometry?MS?,we identified a leucine-rich repeat?LRR?kinase?MdLRRP1?from apple could interact with VmHEP1.What is more,by mean of VIGS,we silenced NbSERK3 which is the homologous gene of MdLRRP1 in N.benthamiana,and the followed transient expression of VmHEP1 in those NbSERK3 gene silenced strains showed significantly less cell necrotic degree.The results showed there was a potential infection-induced promoter on the upstream of gene VmHEP1.Furthermore,kinase MdLRRP1 was a potential interaction target of effector VmHEP1.?3?Pod domain-containing effectors VmPODs contributed to conidiation,H₂O₂ resistance and virulence of V.mali and widely distributed in different fungi.To find a kind of effector containing conserved domain which was still unrevealed,the genome and secretome of V.mali was analyzed.We finally found three candidate effectors?VmPOD1,VmPOD2 and VmPOD3?containing conserved Pod?Peroxiadase?domain.Sequences analysis showed each of the three VmPODs have a signal peptide on N-terminal,VmPOD1 contained two catalase peroxidase domains,VmPOD2 contained one chloroperoxidase domain and VmPOD3 contained one plant peroxidase-like peroxidase domain.The secretion function of VmPODs was confirmed by mean of the yeast invertase secretion test.Futher deletion of each gene did not affect the vegetative growth of V.mali.However,the conidiation and H₂O₂ reisitance of the gene deletion mutants??VmPOD1,?VmPOD2 and?VmPOD3?were significantly reduced.What is more,qRT-PCR showed all of these three VmPODs genes significantly up regulated during early infection,and transient expression of VmPODs in N.benthamiana could reduce the electrolyte leakage in leaves caused by INF1,which implied the blocking of the INF1 induced resistance response.Further,deletion of VmPOD3 resluted in dramatic virulence decline on both twigs and leaves,which indicated VmPODs genes contributed to interaction between V.mali and apple.What is more,phylogenetic analysis demonstrated that VmPODs were not species-specific proteins,homologous proteins of VmPODs widely distributed in different fungi including several important phytopathogenic fungi.The current results provided new insight into the function of this potential and widely distributed candidate effector containing Pod domain.