Pathogenicity Of P.Expansum Effector PeLysM2

Fruit rot caused by P.expansum is one of the most devastating diseases after harvesting.Effector factors play an important role in the interaction between pathogenic bacteria and host.Lys M family proteins are important effectors and play multiple roles in the mutual recognition and signal transduction between pathogenic bacteria and host.The Lys M family proteins have been shown to bind with the fungal cell wall component chitin to inhibit the host immune response induced by fungi,or prevent the degradation of fungal hyphae from chitinase secreted by host.In this study,P.expansum PeLysM2 gene was characterized to elucidate the molecular mechanism interacted between P.expansum and host kiwifruit(A.chinensis),providing theoretical basis for protection for kiwifruits postharvest.In this study,PeLysM2 protein was expressed and purified in Pichia pastoris.PeLysM2 protein was shown to interact with chitin,oligosaccharides and fungal chitin extracted from P.expansum.PeLysM2 was also demonstrated to protect fungal hyphae against the hydrolytic activity of chitinase.We knocked out PeLysM2 gene by homologous recombination.The loss-of-function deletion of PeLysM2 gene did not affect the growth of P.expansum on PDA,but reduced the expansion rate on fruit.Compared with wild-type P.expansum,?PeLysM2 mutant strain significantly enhanced the expression of two resistance marker genes Ach PR1 and Ach CHI during inoculation.Two proteins that interacted with PeLysM2 were indentified by the yeast two hybrid assay.The two proteins are synaptotagmin(Ach Syt L)and the chloroplast transport protein(Ach Tic),respectively.The interaction between PeLysM2 and Ach Syt L or Ach Tic is universal.The interaction between PeLysM2 and Ach Syt L,Ach Tic,Sl Syt5 or SlTic40 was verified with Co-IP assay.Bioinformatics analysis showed that the similarity of protein sequences between Ach Syt L(PSR84718)and Sl Syt5(XP?004235753.1),or between Ach Tic(PSS31813)and SlTic40(XP?004250413.1)were 81.8% or 76.1%,respectively.There are two homologous genes of Ach Syt L in kiwifruit,and the similarity of their amino acid sequence is 93%.Ach Tic also has two homologous genes,and the similarity of their amino acid sequence is 90%.During inoculation,in contrary to the inhibition effect on the expression of kiwifruit Ach Tic gene,?PeLysM2 mutant strain strongly induced the expression of kiwifruit Ach Syt L.By using RNA-interference technique,we silenced the tomato gene Sl Syt5 and SlTic40 in tomato,respectively.Silencing of Sl Syt5 in tomato appeared to accelerate the tomato decomposition after treatment by wild-type P.expansum.Secondly,Sl Syt5 gene played an important role in tomato resistance to infection by P.expansum.SlTic40-RNAi transgenic tomato plants exhibited developmental defects with pale leaves and arrested growth,and produced small and seedless fruits.The SlTic40-RNAi transgenic plants also inhibited the expression of key enzymes in the chlorophyll biosynthesis pathway detected by RT-PCR.