Functional Characterization Of Genes Involved In Wheat Defense Against Stripe Rust Fungus

Wheat stripe rust, caused by the obligate biotrophic fungus Puccinia striiformis f. sp.tritici (Pst), often imposes a tremendous threat to the production of wheat worldwide. Over40stripe rust resistance genes have been identified in wheat, which have been explored to greatlyadvance our understanding of the molecular mechanism of stripe rust resistance. The cloningof disease resistance genes and the dissection of the signal transduction components ofhypersensitive cell death and systemic acquired resistance pathways will greatly amplify thediversity of options available for the application of R genes and provide insights into themechanism of host-pathogen interaction. In this study, two zinc finger protein genes TaLSD1,TaLOL2and a functional homologue of AtNPR1, TaNPR1were characterized in the wheat-stripe rust fungus pathosystem. The main results and conclusions are as follows:1Genetic studies of the Arabidopsis lesion simulating disease mutant1(lsd1) revealed anovel plant-specific zinc finger protein gene family, whose members are significantly relatedto programmed cell death (PCD). A wheat1042-bp homologue of LSD1-type zinc fingerprotein (zf-LSD1), which contains three internally conserved zf-LSD1domains defined byCxxCxRxxLMYxxGASxVxCxxC, was designated as TaLSD1previously in our lab. Theexpression of TaLSD1was differentially induced during incompatible and compatibleinteractions between wheat and Pst, but was suppressed by oxidative stress generated bymethyl viologen (MV). TaLSD1was found to be predominately localized within the nucleusand randomly distributed in the plasma membrane. Transient overexpression assays inNicotiana benthamiana demonstrated that TaLSD1partially inhibited programmed cell deathtriggered by a mouse Bax protein, whereas expression of TaLSD1alone had no influence onthe phenotype of tobacco. Knocking down the expression of TaLSD1through virus-inducedgene silencing (VIGS) increased wheat resistance against Pst accompanied by an enhancedhypersensitive response (HR), an induction of PR1gene expression and a reduction in Psthyphal growth. Our results suggest that TaLSD1functions negatively in regulating the planthypersensitive cell death and is involved in disease resistance of wheat against the stripe rustpathogen.2Because the zf-LSD1domain containing proteins are often essential regulators of celldeath, we further searched the wheat EST database in GeneBank and the wheat cDNA libraries constructed in our lab using the AtLSD1as the initial probe. Another LSD1-typeprotein, which also contains three zf-LSD1domains but is different from TaLSD1, wascloned by in silico cloning and RT-PCR and designated as TaLOL2. TaLOL2shares highsimilarity with rice OsLOL2, which was predicted to encode a protein with179amino acidresidues. TaLOL2transcripts in wheat leaves was induced by the infection of stripe rustpathogens at the early stages. The expression pattern was similar in the incompatible andcompatible interactions between wheat and stripe rust fungus. It was speculated that TaLOL2was involved in the basal resistance of wheat against stripe rust fungus.3The Arabidopsis NPR1protein is a critical regulator of systemic acquired resistance,which mediated signal transduction pathway leading to the induction of pathogenesis-related(PR) genes and enhanced disease resistance. Through in silico cloning, RT-PCR and RACEmethod, a functional homologue of AtNPR1, TaNPR1was cloned, which encodes a protein of542amino acid residues containing an ankyrin repeat domain and a BTB/POZ domain. Theexpression of TaNPR1was significantly up-regulated in the incompatible interaction at12hours post-inoculation (hpi), suggesting that TaNPR1is likely involved in resistance of wheatagainst stripe rust fungus, at48hpi, the expression of TaNPR1reached another peak duringboth incompatible and compatible interaction. Knocking down TaNPR1expression throughVIGS enhanced the susceptibility of wheat against the avirulent race of Pst with enlargednecrotic area per infection site and sporulation. Meanwhile, the hyphal lengths of Pst weresignificantly longer compared with control treatment. These results suggest that TaNPR1functions positively in regulating wheat defense response to stripe rust pathogens.