Functional Analysis Of Bsr1 Against Barley Stripe Mosaic Virus In Brachypodium Distachyon

Viral diseases caused a decline in quality and yield in major crops. Discovering viral resistance genes is critical for crop virus-resistance breeding and plant-pathogen interactions studying. Barley stripe mosaic virus (BSMV) is a single strand (+) RNA virus with a tripartite genome and causes a serious world-wide disease on barley and other cereals. However, very little is known about the host resistance against BSMV in crops. In the lab conditions, BSMV has the ability to infect Nicotiana benthamiana, Brachypodium distachyon and Chenopodium Amaranticolor for studying of BSMV pathogenesis and virus movement. B. distachyon is a member of the Poaceae subfamily Pooideae and has emerged as a model species for the study of cool season cereal crops.Brachypodium inbred line Bd21 showed highly susceptible to BSMV North Dakota 18 (ND18) strain, but inbred line Bd3-1 showed highly resistant to ND18. The barley stripe mosaic virus resistance 1 (Bsr1), a CC-NB-LRR gene, is responsible for the resistance of Bd3-1. Analysis of the function of Bsrl would benefit the study of the molecular mechanism of BSMV resistance and gives clues to monocot plants virus resistance. The major results are listed as following:1. The Bsrl gene overexpression construct pMBsrl driven by double CaMV 35S promoter, and the Bsrl gene complementary construct pCBsrl under native promoter were used for transformation of BSMV ND18 susceptible Brachypodium line Bd21-3. The results showed that Bsrl overexpression transgenic Bd21-3 plants performed BSMV resistance against ND18 strain, indicating that Bsrl is the key gene for BSMV resistance in Brachypodium line Bd3-1.2. To test whether the BSMV resistance gene Bsrl from Brachypodium works in barley and wheat, the complimentary construct pCBsrl was transformed into barley cultivar "Golden Promise" and wheat cultivar "Ke Nong 199" (KN199), two susceptible cultivars to BSMV ND18 strain. PCR results indicated that the Bsrl gene was successfully integrated into Golden Promise" and "Ke Nong 199" genomes. The transgenic barley showed highly resistance to BSMV strain ND18, indicating that the Brachypodium gene Bsrl functions in barley and could be used for barley viral resistance improvement.3. Co-expression of the BSMV strain ND18 amplicons and Bsrl proteins elicited cell death in N. benthamiana, but ND18 amplicons and the susceptible allele bsrl does not. Based on the amino acid sequences diversity between Bsrl and bsrl,10 domain-swaped chimeric constructs were cloned into binary vector pMDC32 for functional characterization of the Bsrl/bsrl domains in N. benthamiana. These constructs were transiently coexpressed with the ND18 amplicons in N. benthamiana leaves to detect whether a necrotic lesion was induced. Only one chimeric construct bNBLT containing bsrl CC-NB-ARC domain and Bsrl LRR domain and C-terminal induced cell death phenotype as that elicited by Bsrl, indicating that bsrl has a functional CC-NB-ARC domain and the intact LRR domain and the C terminal fragment of Bsrl are necessary for the viral resistance function against BSMV ND18.4. RNA-seq analyses were performed on Bd3-1 at 1,6, and 14 days post inoculation (dpi) with BSMV strains ND18 (avirulent) and ND18 mutant (TGB1 double mutant TGB1R390K,T392K, virulent) for characterizing the genes involved in salicylic acid (SA) signaling pathway and jasmonic acid (JA) and ethylene (ET) signaling pathway. The results showed that most of SA signaling components such as pathogenesis-related (PR) genes and WRKY transcription factors were not induced or suppressed by ND18 infection, and AOX1B, WKRY18 and WRKY70 were up-regulated at 14 dpi. However, ACO1 and 2 ethylene-response factor (ERF) genes involved in JA/ET pathway were up-regulated at 1 dpi, and 4 ERF genes were induced at 14 dpi. In compatible interactions between Bd3-1 and ND18 mutant, most of SA and JA/ET signaling components showed no expression or suppressed at 1 dpi. At 6 dpi and 14 dpi, genes involved in salicylic acid (SA) signaling pathway was significantly up-regulated. However, except for LOX and ERF9, the genes in JA/ET pathway were not expressed or down-regulated. In the incompatible interactions between Bd3-1 and ND18, SA signaling was suppressed and part of JA7ET pathway was involved. By contrast, in the compatible interaction between Bd3-1 and ND18 mutant, SA signaling pathway were activated.