Preliminery Analysis Of Phytophthora Infestans RXLR Effector Genes Upregulated During Infection And P. Parasitica Effector Gene PPE4

Phytophthora,a genus of Oomycetes,is well-known as"the plant destroyer".P.infestans is the most notorious pathogen and causes potato late blight.It was the causal agent of"Irish potato famine",and still causes huge economic losses to modern potato production.And P.parasitica,being phylogenetically close to P.infestans,not only triggers tobacco black shank but also infects a wide range of plants.Both P.parasitica and P.infestans are hemibiotrophic pathogens that require suppression of plant cell death to maintain biotrophy at the early infection stages,and induction of cell death to facilitate the transition into necrotrophy.Therefore,the manipulation of host plant cell death is particularly important during infection process.RXLR effectors are a category of secreted virulence factors unique to Oomycetes,which are able to enter plant cells and exerts virulence function.There are hundreds of RXLR effector genes in the genomes of P.infestans and P.parasitica,but the function of only a few of them have been partially resolved.An understanding of the biological function of RXLR effectors and the identification of their host targets are conducive to the illumination of the pathogenic mechanisms of Phytophthora,the mining of new resistance genes,and the development of effective disease-control strategies.In this study,the function of candidate RXLR effector genes from P.infestans and P.parasitica were analyzed by Agrobacterium tumefaciens-mediated transient expression,including their subcellular localization,ability to induce or suppress cell death,and contribution to pathogen infection.Furthermore,real-time quantitative PCR,polyethylene glycol?PEG?-CaCl₂-mediated protoplast transformation,VIGS and polymorphism analysis were performed to explore the virulence function and the cell death-inducing activity of a P.parasitica RXLR effector gene PpE4.Finally,the function of PpAvh3a,an effector gene of P.parasitica which shows conserved synteny with P.infestans Avr3a,was analyzed.The main results are as follows:1.Transient expression analysis of 50 candidate P.infestans RXLR effector genes up-regulated during infection stage showed that 47 effector genes could suppress cell death triggered by PAMPs or effectors?PTI or ETI?;one?E73?could induce cell death on Nicotiana benthamiana leaves,and one?E83?caused slight yellowing of leaves.Subcellular localization assays indicated that the highly diversified RXLR effectors suppress plant immunity by localization in various subcellular compartments such as plant cytoplasm,plasma membrane,endoplasmic reticulum,vesicular structure,and nucleus.2.Several Avrblb2 homologous proteins from P.parasitica and P.infestans are relatively conserved,mainly localized in the plant nucleus,and particularly accumulated in nucleolus,nucleoplasm,or nuclear speckles in cell nucleus.In addition,transient expression of P.infestans E47 and E49 and P.parasitica PpE2 and PpE3 triggered cell death in specific tobaccos,suggesting the possible recognition by potential resistance genes.Transient expression of E41,E47 and E49 rendered N.benthamiana more susceptible to P.parasitica,and the transgenic Arabidopsis thaliana expressing E47 and E49 showed dwarf and abnormal growth phenotypes,indicating that they are all virulence factors of P.infestans.3.PpE4 is a P.parasitica RXLR effector gene significantly up-regulated during early infection stages.Live cell imaging of P.parasitica transformants expressing a full-length PpE4?E4FL?-mCherry protein showed that it accumulated in the extra-haustorial matrix?EHMx?after secreted from haustoria,and translocated into plant cells.Silencing of PpE4 in P.parasitica resulted in significantly attenuated pathogenicity,and its transient expression in N.benthamiana in turn restored the pathogenicity of the PpE4-silenced lines.Furthermore,the expression of PpE4 in N.benthamiana and Arabidopsis consistently enhanced plant susceptibility to P.parasitica.These results indicate that PpE4 plays a critical virulence function during P.parasitica infection.4.PpE4 triggered broad-spectrum cell death in various plants including tobacco,tomato,potato,and Arabidopsis.Cell death assay after VIGS-mediated transient silencing of PTI or ETI pathway related genes in N.benthamiana showed that silencing of HSP90,NPK or SGT1significantly attenuated or abolished PpE4-induced cell death.This suggested that PpE4-induced cell death was likely a result of recognization by the plant immune system.Polymorphism analysis revealed that there are 4 PpE4 alleles in P.parasitica population and PpE4 is absent in genome of some strains,and its variation in different strains is associated with hosts and collection regions.Mutation of six amino acids in E4D led to loss of the cell death-inducing activity,indicating that PpE4 might escape recognition through genetic variation.5.P.parasitica candidate RXLR effector PpAvh3a?Avirulence homolog of Avr3a?shares little sequence similarity to P.infestans Avr3a and Hyaloperonospora arabidopsidis ATR1although they are syntenic.PpAvh3a was extremely lowly expressed during infection.It neither induced cell death nor suppressed PTI nor ETI-related cell death in plants,nor contributed to P.parasitica infection.These results indicated that PpAvh3a does not have virulence or avirulence functions as that of PiAvr3a.Although PpAvh3a did not trigger hypersensitive response,its overexpression in P.parasitica led to the attenuation of pathogen pathogenicity.In summary,several candidate RXLR effector genes with virulence function were identified from P.infestans and P.parasitica,and the virulence function and cell death-inducing activity of P.parasitica PpE4 were studed in depth.These results laid a solid foundation for screening unknown resistance genes and analyzing the pathogenic mechanism of Phytophthora.