Analysis Of The Resistance Mechanism Of Brachypodium Distachyon Against Heterodera Avenae Based On Gene Expression Profile

Heterodera avenae is one of the most important plant parasitic nematodes on cereal crops, which causes great yield reduction on wheat production in the world. Brachypodium distachyon is an annual temperate grass with dwarf strains, small genome, simple growth conditions, short life cycle and an efficient transformation, which make it as an ideal material for model research. In addition, genome sequencing and annotation of B. distachyon (genotype Bd21) have been completed and it has a close genetic relationship to wheat. In this study, the process of H. avenae penetrating and the cell morphological and physiological variations of B. distachyon were observed and the transcriptional variations of differentially expressed genes of B. distachyon during different periods after inoculation with H. avenae were also explored by digital gene expression profiling. A series of candidate resistance genes were screened to reveal probable resistance mechanism of B. distachyon against H. avenae. Main results were as follows:(1) Based on comparison of the attraction to different varieties of B. distachyon and wheat with H. avenae juveniles (J2s), all kinds of root tips were able to attract J2s, but the attraction number was significantly different. The numbers of J2s around root tip of B.distachyon PI219961 were almost 113 per plant after 72 h of inoculation, which was the largest among all the varieties.95 J2s were around the root tip of B.distachyon PI254867 after 96 h of inoculation, and 30 J2s were around the root tip of Zhengmai 9023 after 144 h of inoculation.(2) The process of H. avenae penetrating the roots of B. distachyon showed that J2s mainly distributed and feed along the vascular bundle in the root cortex after penetrating from the main root. A small number of nematode successfully invaded into the roots of B. distachyon and only J2 stage was observed which indicated that the nematodes could not complete the entire life cycle.(3) Amounts of necrotic cells were discovered in elongation and meristematic zones of root tips by Typan blue staining analysis. It may be caused by hypersensitive reaction (HR) when J2 invaded into the roots. Initial syncytial cells (ISC) occurred but no further syncytium developed in the radical root of B. distachyon. Necrotic cells subsequently happened around the syncytium. The activities of defense-related enzymes such as peroxidase (POD) and phenylalanine ammonia-lyase (PAL) in the roots of B. distachyon increased significantly after inoculation with H. avenae. DAB staining showed that hydrogen peroxide (H2O2) was accumulated in the elongation zone as well as the meristematic zone of the root tips of B. distachyon, which indicated that reactive oxygen species (ROS) was involved in the resistance reaction.(4) Through the analysis of expression profiling based on next-generation sequencing, gene expression data of B. distachyon roots in seedling stage after inoculated with J2s at 24 hours and 6 days and the control groups were obtained. The comparison of expressed genes at different inoculation times showed that there were 1593 up-regulated genes and 1088 down-regulated genes at 24 hours after inoculation, while 831 up-regulated genes and 2047 down-regulated genes were found at 6 days after inoculation. Gene Ontology analysis showed that the differentially regulated genes mainly involved the lignin metabolic process. KEGG pathway analysis indicated that the significant metabolic pathways involved phenylalanine metabolic pathway, biosynthesis of secondary metabolites, plant-pathogen interaction, metabolic pathways, and plant hormone signal transduction. A series of candidate resistance-related genes such as cytochrome P450, peroxidase, L-ascorbate oxidase, universal stress protein A, homeobox-leucine zipper protein HOX21, agglutinin isolectin 3, and mixed-linked glucan synthase 3 were screened.(5) The resistance mechanism of B. distachyon against H. avenae mainly depended on restriction the invasion number of nematode and the development of J2s in the root tissue after penetration by resistance genes, defense-related enzymes, signal transformation, hypersensitive reaction (HR), reactive oxygen species (ROS). The results will be helpful to the research on nematode-plant interaction between H. avenae and wheat.