Functional Analysis Of PINP1 In Plant Small RNA Biogenesis And Defense Responses

As one of the most important food crop and oil crops,the soybean(Glycine max)plays an important role in the agricultural industry.However,it is susceptible to many pests and diseases infestation in the growth process.These pests and diseases not only affect their growth,but also affect the yield and quality of soybeans.For example,root rot of soybean caused by Phytophthora sojae can cause loss of billions per year worldwide.P.sojae secretes a large number of RxLR effectors to enter plant cells to disturb plant immunity and promote infection.Based on the previous work of the P.sojae effector PSR1 and its target PINP1,PINP1 and its homologous proteins were further studied.First,the RNA-seq analysis of PSR1-expressing plants and PINP1-silenced plants was performed.Then we cloned GmPINP1 a,GmPINP1b and OsPINP1 genes in soybean and rice,constructed related vectors,and performed subcellular localization and protein interaction analysis.Finally OsPINP1 was used as bait protein to screen the interaction proteins in the cDNA library of rice.The main results are as follows:1.By transcriptomic analysis of PSR1-expressing plants and PINP1-silenced plants,a large number of differentially expressed genes involved in cell part,binding and metabolic processes were obtained;The results of the alternative splicing analysis showed that a large number of intron retention events occurred.In both lines,differentially expressed genes and genes that have intron retention events are highly overlapping.2.Bioinformatics analysis revealed that genes with intron retention events contain multiple genes involved in small RNA biosynthesis and jasmonic acid signaling pathways.This indicates that PSR1 binds to the PINP1 protein and thereby disrupts the biosynthesis of small RNA and the pathogenic response pathway regulated by jasmonic acid.3.GmPINP1 a,GmPINP1b and OsPINP1 with the full CDS lengths of 3819 bp,3816 bp and 3864 bp were cloned by PCR amplification respectively.The subcellular localization showed that all three proteins localized in the nucleus by transient expression in Nicotiana benthamiana leaves.4.The interaction between all three homologous proteins of PINP1 and PSR1 were verified by Y2 H,bimolecular fluorescence complementation(BiFC)and Co-Immunoprecipitation(Co-IP)assays.5.Using the yeast two-hybrid technique,some interaction proteins of OsPINP1 in rice were screened,and some potential interaction targets were underway.