Functional Analysis Of Effector PsCRN78 From Phytophthora Sojae

Phytophthora sojae is a plant pathogen that causes stem and root rot of the soybean. Each year in the most soybean-growing regions of the world, millions of dollars are lost. It shares similar growth habits with fungi, but it is evolutionarily closed to diatom and blue algae, therefore is classified in kingdom Stramenopila, Phylum, Oomycota. Phytophthora spp. formed the pathogenic mechanism which is different with these phytopathogenic fungi. And most fungicides show no control effect to plant diseases caused by P. spp. Therefore, looking for an effective strategy to control against the pathogens has been being the primary task of researchers. To achieve this level of host colonization, plant and pathogens secrete an arsenal of molecules, termed effector, that interfere with host immune pathways and enable host colonization. It was reported that CRN gene family was cytoplasmic effectors of oomycetes. Since the proteins of Phytophthora infestans could induce cell death in Nicotiana benthaminana, they were termed as crinkler and necrosis protein (CRN). However little is known about their functions and mechanism.In this study, we detected the transcription of four genes (PsCRN78, Avr1k, PsISO) in P.sojae and SGT1 gene in soybean during interaction between soybean and P.sojae. Transcriptional levels of all the tested genes in P.sojae were up-regulated after infection. The SGT1 gene was highly induced upon P. sojae infection in two soybean cultivars that confer different resistant levels. These results suggest that PsCRN78, Avr1k, PsISO and SGT1 might play a role in the interaction between soybean and P.sojae.Here, we also analyzed the gene sequence of PsCRN78, an effector of P. sojae. We found that PsCRN78 contains a classic FLAK translocation motif and a kinase motif, so PsCRN78 might be an effector which can be translocated to the host cells to regulate the host defense through the kinase activation. In order to valid the function of the N-terminal assumed signal peptide, the result in yeast has been demonstrated that it can be secreted out of the yeast cells. On the other hand, PsCRN78 can be accumulated around the haustorium after infection. These results indicate that PsCRN78 is a secreted effector.The function of PsCRN78 was analysised. We used the PEG-mediated gene transformantion technology of P.sojae and obtained two silence transformants, in which expression of PsCRN78 gene is impaired. Compare to wild type, the two silence transformants reduced the pathogenicity of P. sojae. By overexpressing of PsCRN78 in N. benthamiana and Arabidopsis, we found that it reduced plant resistance against pathogens. The results suggest a role for PsCRN78 in the suppression of host defense responses. We also found that PsCRN78 localizes to the host plasma membrane and nucleus. The plasma membrane localization is required for the PsCRN78 virulence activities. To determine if the kinase activity is essential for PsCRN78 to supress host defence, we obtained a mutant PsCRN78-EDRm (substitutionsin kinase activity site). By tumefaciens-mediated transient expression of PsCRN78-EDRm in N.benthaminana, PsCRN78 showed no localization change, but lost of its ability to suppress host defense. These results indicated that the virulence of PsCRN78 is dependent on its kinase activity.We have studied the influence of PsCRN78 for the P.sojae virulence and plant resistance, and the link between its localization and function. Overall, our results indicate that PsCRN78 is a secreted effector, and primarily elucidated the mechanisms underlying the PsCRN78 virulence activities. These results will better our understanding of CRN effectors.