Identification And Functional Analysis Of TaMlo1/2/5and TaLoL2in Wheat Infected By Stripe Rust Fungus

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici, which is one of worldwidepopular wheat fungal diseases. Domestic and foreign research and practices have proved thatbreeding and rational use of resistant varieties is the most secure, economic and effectivemethod to control stripe rust. Both cloning and functional characterization of resistance genesand deep analysis of the molecular mechanisms of interaction between wheat and stripe rusthave important theoretical significance for disease resistance breeding. In this study, wecloned three genes designated TaMlo1, TaMlo2and TaMlo5which may be functionallyrelated with calcium signaling protein and involved in hypersensitive cell death signalingpathway and performed preliminary functional analysis of these three genes. In addition, wefurther functionally characterized a wheat zinc-finger protein gene TaLOL2which had beenpreviously cloned in our laboratory.(1) Based on the Mlo gene sequences deposited in Genbank from wheat cultivar ’ChinaSpring’, we cloned three wheat genes TaMlo1, TaMlo2and TaMlo5by RT-PCR from wheatcultivar ’Sunwon11’. Sequence analysis indicated the three genes encoding534,499and492amino acids and all three encoded proteins containing seven trans-membrane domain which isunique for the Mlo family in plants. Real-time RT-PCR assays showed that TaMlo1, TaMlo2and TaMlo5exhibited up-regulation expression tendency in both compatible and incompatibleinteraction of wheat and stripe rust fungus. However, the expression level was relativelyhigher in incompatible interaction than that of compatible interaction. After four exogenoushormones treatment, both TaMlo1and TaMlo2had some degree of up-regulation, whereasETH does not have effect on the expression of TaMlo5. The expression of TaMlo1, TaMlo2and TaMlo5showed up-regulated trend in different degrees after wound, cold and methylviologen treatments. Expression levels of TaMlo1, TaMlo2and TaMlo5in root, stem and leafof wheat are similar. Subcellular localization assays showed that TaMlo1, TaMlo2andTaMlo5were localized in the epidermal cell membrane of tobacco. Yeast two-hybrid assaysshowed that TaMlo1, TaMlo2and TaMlo5can directly interact with TaCaM4under appliedcalcium ions, respectively. Agrobacterium-mediated tobacco transient expression analysisshowed that TaMlo1, TaMlo2and TaMlo5can significantly inhibit the BAX protein triggeredprogrammed cell death (BT-PCD), while TaMlo2can delay the time of the PCD. Furthermore, knocking down expression of TaMlo1and TaMlo5by virus induced gene silencing enhancedthe susceptibility of wheat cv. Suwon11to an avirulent race CYR23. These results imply thatTaMlo1and TaMlo5play roles in the early stage of wheat-stripe rust fungus interaction and instress tolerance as a negative regulator of PCD.(2) We further functionally characterized a wheat zinc-finger protein gene TaLOL2which had been previously studied in our laboratory. Subcellular localization assays revealedthat TaLOL2was localized in the nucleus of onion epidermal cells. Real-time quantitativeanalysis showed that ABA can significantly induce TaLOL2expression, while other planthormones and abiotic stresses had no effect on transcript level of TaLOL2. Knocking downTaLOL2expression by virus induced gene silencing enhanced the susceptibility of wheat cv.Suwon11to an avirulent race CYR23. The results suggest that TaLOL2may be involved inwheat defense against stripe rust fungus as a positive regulator of PCD.