Characterization And Functional Analyses Of BdWRKY Transcription Factor In The Brachypodium Distachyon-rust Fungus Interactions

Wheat stripe rust is one of the major diseases that affect the safety of wheat production in China.The rapid virulence variation of Pst results in“resistance loss”of wheat cultivars,and leads to the frequent epidemic of the disease.Thus,it is significant for durable control of this disease to reveal the molecular mechanism of wheat resistance to stripe rust and develop new control strategies.WRKY TFs,a superfamily of plant transcription factors?TFs?,play important roles in plant defense against pathogens through regulating expression of the defense related genes.However,little is known about the key WRKY TFs and their function on wheat resistance to stripe rust.Because of the large genome and low efficiency of genetic transformation,the wheat genome is not conducive to the functional verification of the rust resistant gene and its complete genetic characteristics.,utilizing the molecular mechanism of model plants to study the interaction between rust fungi and model plants is of great significance for revealing the mechanism of stripe rust resistance in wheat.Thus,we plan to initially screen the key WRKY TFs which contribute to host resistance during the Brachypodium distachyon?Bd?–Puccinia brachypodii?Pb?interaction,and study their histological characteristics and the gene expression profiles using RNAi.Our results have characterized the roles of WRKY TFs in Bd-Pb interaction,revealed the molecular mechanism underlying Bd-Pb mediated by WRKY TF.Taken together,our results will provide theoretical basis for developing new strategies to durable control of wheat stripe rust.The main results are as follows:1. The infection process of Pb in leaves of Bd21-3 was investigated.After inoculation on the Brachypodium distachyon inbred line Bd21-3,the Pb isolates F-CO and K-Ki infected plant leaves and formed a substomatal vesicle?SV?at 12 hai,then formed a haustorial mother cell?HMC?.At 18 hpi,HMC invaded plant cells to form haustoria.At 24 hpi,the pathogen infecting hyphae?IH?began to branch that lead to the formation of secondary hyphae?SH?.At 48 hpi,the pathogen produce more branches.At 72 hpi,secondary hyphae developed further to form small microcolony.At 120 hpi,SH formed additional branched infection hyphae,which constituted microcolony in plant tissue.The growth rate of F-CO in the leaves is faster than that of H-KI.The microcolony expansion area of F-CO was obviously larger than that of H-KI.No significant difference in the number of HMC and haustoria of aspirator between F-CO and H-KI was observed at the initial stage of colonization by the pathogen.However,the number of IH,HMC and haustoria of Bd-Pb interaction were significantly more than those of wheat-stripe rust interaction.This study confirmed that F-CO,H-Ki and Bd21-3 were all compatible and did not produce H₂O₂.2. To find out the BdWRKY transcription factors involved in the Bd-Pb interaction,we have analyzed the expression of 7 BdWRKY TFs?BdWRKY31,-36,-38,-60,-62,-68,-78?by q RT-PCR.Their expression were up-regulated from the 24 hpi to 72 hpi,indicating that they are involved in the interaction between Bd21-3 and F-CO or H-KI.Bioinformatics analysis showed that all the proteins contained the conserved WRKY domain and typical Zine finger region,which were similar to other WRKY of the grass family.Five BdWRKY TFs?BdWRKY31,-38,-60,-68,-78?from Bd's c DNA were cloned from Bd21-3.Subcellular localization of the five WRKY proteins in the tobacco cells showed that all of them were located at nucleus,indicating that their WRKY domain binding with target genes in plant nuclei to regulate the resistant/susceptible process of Bd.3. In this study,the transgenic plants of seven BdWRKY TFs were obtained using theAgrobacterium mediated genetic transformation system of Bd successfully established in our laboratory.At least three lines of the fertile transgenic plants for each gene were obtained.The average transformation efficiency was 36%.PCR detected that the number of positive plants for the BdWRKY31,-32,-38,-60,-62,-68 and-78 was 6,3,10,1,20,21and 12,respectively.4. Based on the 10-level differential system of Brachypodium hirsutum established in this study,the phenotypes of T3 families with positive plants were tested and shown in Table 4-8.The silencing efficiency of the mutants BdWRKY31R-4,38R-6 and 60R-2 was 89%,73%and50%,respectively.The mutants of these three genes showed medium resistance to F-CO.Suggesting that BdWRKY31,BdWRKY38 and BdWRKY60 play negative roles in defense responses of Brachypodium distachyon to F-CO.