Validation Of Phased Small Interfering RNA Genes And Their Expression Analyses In Response To Botrytis Cinerea Infection In Solanum Lycopersicum

Botrytis is a plant disease caused by ?Botrytis cinerea‘,which is characterized by widely spread and hard to control.This plant disease causes huge loss to yield of vegetable plants every year.The objective of this study was 1),to identifying phasi RNA genes which can response to Botrytis in tomato（Solanum lycopersicum L.）;2),to determine the transcription of the Botrytis responsive phasiRNA genes before,during or after Botrytis cinerea infection in tomato.Hopefully,we can identify some Botrytis responsive phasiRNA genes and these can be used as marker to selecting Botrytis resistant tomato cultivars.The phasi RNA is a kind of 21-24 nt siRNA,which is generated from the mRNA of PHAS gene catalyzed by miRNA,RDR6,and DCL.Previously studies suggested that phasi RNA play role in many physiological pathways through regulating its downstream transcriptional factors.In order to validate and study phasiRNA in tomatoes,molecular biology and bioinformatics method were used in this study.The main results are as follows:1.34 out of 90 tomato PHAS candidate genes were validated using RT-PCR.Among the validated genes,21 PHAS were located in the intragenic loci,6 were partially overlapped with 5′-or 3′-end of known genes,and the remaining 7 PHAS were located in the new loci.2.sRNA-seq data revealed that 2 phasi RNAs derived from 2 PHAS（Sly-PHAS04 and Sly-PHAS26）and 12 additional phasiRNAs derived from 8 PHAS genes were downregulated and upregulated in Botrytis cinerea-infected leaves,respectively.3.Of these 14 phasiRNAs,six were confirmed to express differently in B.cinerea-infected leaves compared with the control leaves in tomato.Three coding genes of glycosyl-phosphatidyl inositol-anchored protein（Solyc01g065530.2.1）,ribonuclease 3-like protein 3（Solyc11g008540.1.1）,and MYB transcription factor（Solyc03g113620.2.1）were negatively regulated by their corresponding phasiRNAs in B.cinerea-infected leaves.This result suggests these phasiRNAs may regulate these target genes to respond to B.cinerea infection in tomato leaves.4.Sly-PHAS15 gene Clone and instantaneous expression technology were employed to further validate Sly-PHAS15₃042（-）response to B.cinerea infection in tomato leaves.The results showed that Sly-PHAS15₃042（-）was significantly upregulated in Sly-PHAS15 overexpression tomato leaves,which tends to be infected by B.cinerea more easily.This suggests that Sly-PHAS15 can product Sly-PHAS15₃042（-）and they may play a positive role in the process of B.cinerea infecting tomato leaves.5.In this study,we compared the transcription level of Sly-PHAS15 and phasiRNA Sly-PHAS₃042（-）in different organs,such as root,steam,leaf,flower,and fruit of tomato（cv.?Microtom‘）.Our results indicated that the transcription of Sly-PHAS15 is significant higher in leaf and flower,while significant lower in root compared to steam and fruit.For Sly-PHAS₃042（-）,its expression is higher in steam and flower,while lower in fruit and almost undetectable in root.