Genome Analysis Of Non-host Resistance Of Brachypodium Distachyon To Stripe Rust And Functional Characterization Of BdNAC Transcription Factors Family

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most destructive diseases of wheat.To control stripe rust,the disease-resistant varieties were cultivated in current wheat production.However,most of resistance genes in wheat have beenovercome due to the virulent variation.So new and durable rust resistance genes resources need be explored and tilized in wheat production.Nonhost disease resistance(NHR)is an entire plant species toall genetic variant of a nonadapted pathogen species.Besides,NHR is durable and strong stability for disease resistance.Thus,the utilization of NHR gene resources for controlling stripe rust is very important in wheat production.In this study,using microarray techniques and available complete B.distachyon genome sequences,we analyze the gene expressions in B.distachyon after Pst and Puccinia.brachypodii inoculation.Base on the microarray results,we did research in expressions and functions of NAC transcription factors in B.distachyon after Pst and P.brachypodii inoculation.This study is significance to understand the mechanism of NHR,and explored new NHR genes resources.1.Genome analysis of non-host resistance of Brachypodium distachyon to stripe rustThe phenotypes of Brachypodium distachyon(B.distachyon)infected by Puccinia brachypodii and Puccinia striiformis f.sp.tritici(Pst)were different.On the host plants,massive uredia were produced on the leaf surface 14 days after inoculation(dai).In contrast,no visible symptoms were observed on wheat leaves inoculated with Pst.Using microarray techniques and available complete B.distachyon genome sequences,we found 889 genes and 524 genes were changed the expressions(log2≥1 or ≤-1,p-value≤0.05)in B.distachyon after Pst and P.brachypodii inoculation,respectively.There were 128 genes changed expression in both interactions.q RT-PCR was used to validate the microarray results.The validation results were consistent with the gene expression patterns identified by microarray results.We found these genes were strongly induced in the early infection stage.The results indicated that expressions of these genes were fast and sensiritive in B.distachyon– Pst interactionsGene Ontology functional analysis showed that there was overlap between host andnon-host defence mechanisms as based defence.However,we found some specific the GO enrichment of Pst-B.distachyon.The significant GO terms were response to ‘response to jasmonic acid’,‘plant-type hypersensitive response’ and‘regulation of innate immune response’.In particular,plant cell wall and econdary metabolites played important roles in resistance Pst.All these data showed that there were intricate mechanisms to regulate the NHR in B.distachyon after Pst inoculation.2.Functional characterization of NAC transcription factors familyWe selected 100 NAC genes from B.distachyon genome to investigate gene structures and phylogenies.We performed the comparative analysis of NAC genes in B.distachyon,rice and Arabidopsis.23 B.distachyon NAC genes were distinguished from other plant NAC members,and 14 of which were cloned.The Phylogenetic tree result showed that these 18 NAC genes divided in CladeⅡ.We analyzed the expressions of these NAC genes by qRT-PCR.There were different expressions with these NAC genes in 4 tissues(leaf,root,stem and spike),especially in leaf and root.The expression of 8 NAC genes were inhibited in 120 hpi when B.distachyon after P.brachypodii inoculation.4 NAC genes were up-regulated in the early stage.Besides,there were 11 NAC were up-regulated when B.distachyon after Pst inoculation,9 of which reached the peak at 24 hpi.The results indicated that of these genes played different roles in plants resistance to rust fungi.We also used RNAi to further analyze the functions of these selected NAC genes.And we got 10 NAC genes RNAi transgenetic seedling.We found there were not obviously distinguish of growth phenotypes,except knockdown of BNAC65 showed B.distachyon was short and the spike of knockdown of BdNAC67 lines were bigger than wide types(WT).The results indicated that NAC genes involving in the growth of plants.We silenced these NAC genes to explore their functions in plant and fungi interactions.When BNAC95 was knockdown,the phenotypes of B.distachyon infected by P.brachypodii were different.The infection hyphae of P.brachypodii did not forme uredia in B.distachyon leaf,and there were black spots.Silencing of BNAC37 showed that there were not obviously phenotypes when B.distachyon infected by Pst.Microscopically,P.brachypodii formed haustorium mother cell(HMC)at the tip of the infection hypha at 24 hour post inoculation.But Pst were arrested to the formation of HMC and failed to develop haustoria.Besides,we found BNAC32 were induced when B.distachyon infected by Fusarium graminearum,which highly expressed in spike.When knockdown of BNAC32,B.distachyon was more sensitive to Fusarium graminearum.The results indicated that NAC genes may play different roles in resisting diseases.