Preliminary Functional Analysis Of RXLR Effector Genes Pi04196 And Pi09732 From Phytophthora Infestans

Potato is the fourth largest food crop in the world,but its sustainable production is seriously threatened by late blight which is caused by Phytophthora infestans.P.infestans belongs to oomycete and secretes numerous extracellular and cytoplasmic effectors,according to whether they are translocated into host plant cells during plant infection.RXLR effectors are the most widely studied oomycete cytoplasmic effectors.P.infestans encodes large number of RXLR effectors to effectively modulate host cell physiology and function to facilitate plant infection.In this research,two candidate RXLR effector genes Pi04196 and Pi09732,which were expessed at early P.infestans infection according to previous RNA-seq data,were focused for their expression pattern,sequence conservation,subcellular localization of their encoded proteins,virulence function.The results have been obtained as follows:The expression P.infestans RXLR effector gene Pi04196 is not detectable in vegetative mycelia while highly up-regulated at early infection stage(6 hours post inoculation,hpi)as revealed by quantitative RT-PCR assays.Pi04196 is highly conserved with no sequence variation detected among 14 P.infestans strains with different genetic background by PCR sequence amplification and comparison assays.Confocal microscopy analysis showed that Pi04196-GFP fusion protein was mainly co-localized with PM marker at plasma membrane in N.benthamiana cells,with small amount of distribution in the nuclear membrane and cytoplasm.In A.thaliana protoplasts,Pi04196-GFP were consistently accumulated at the plasma membrane.Agrobaterium tumefaciens-mediated transient expression of Pi04196 in Nicotiana benthamiana rendered significantly the plants susceptible to infection by P.infestans.Stable expression of Pi04196-GFP in Arabidopsis thaliana rendered the plants grew slowly,with delayed bolting and flowering,and significant shorter root compared to the wild type Col-0and control plants.Furthermore,Pi04196-GFP-expressing A.thaliana plants were more susceptible to Phytophthora parasitica infection.Transient expression of Pi04196 in several potato cultivars through Agrobacterium-mediated approach showed that Pi04196 triggered specific hypersensitive response(HR)on cultivar Longshu 12.Further analyses showed that Pi04196 did not induce HR on potato differential cultivars Ma R1-Ma R11.These results suggested Pi04196 is a novel avirulence gene and Longshu 12 carries a novel late blight resistance gene.Quantitative RT-PCR analyses showed that RXLR effector gene Pi09732 was expressed in vegetative mycelia at low levels while highly up-regulated during infection,peaking at 6hpi.Sucellular localization assays consistently showed that Pi09732 is mainly localized on the plasma membraneof N.benthamiana cells and A.thaliana protoplasts,though it’s weakly accumulated in the nucleolus.Pi09732 significantly promoted the infection of P.infestans when transiently expressed in N.benthamiana.Stable expression of Pi09732 in A.thaliana had no obviously morphological influence,but significantly enhanced infection and colonization by P.parasitica.In addition,Pi09732 delayed INF1-induced cell death when co-expressed in N.benthamiana leaves,suggesting that Pi09732 contributed virulence of P.infestans by suppression of INF1-induced PTI.