| Blumeria graminis f. sp tritici and Fusarium graminearum are among the most devastating fungal pathogens in wheat (Triticum aestivum L.), causing powdery mildew and Fusarium head blight diseases, respectively. Both pathogens constantly experience population changes, and this brings practical difficulties to disease managements. Effective approaches have been proposed to be the application of resistance genes targarting specific isolates or populations of pathogens and additionally, the integration of broad-spectrum resistance mechanisms into wheat germplasm. In this study, therefore, the author cloned a predicted wheat gene designated as TaRGA, which belongs to the NBS-LRR class of resistance genes and hypothetically plays a role in specific resistance to powdery mildew. This hypothesis was examined by analyzing the effects of TaRGA on the powdery mildew disease and wheat defense responses. With respect to broad-spectrum resistance, Hpal is a harpin protein produced by the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae and induces resistance to different diseases in a variety of plants. Thus, the author performed a transgenic protocol, by which the functional fragment Hpal 1042 from the Hpal sequence was introduced into the wheat genome, and the subsequent effects on the crop resistance to powdery mildew and Fusarium head blight were investigated.1. Cloning and expression analysis of the putative NBS-LRR gene TaRGAThe degenerate primers were designed according to the amino acid conserved regions of the konwn disease resistance genes and subsequently were used to amplify the resistance gene analog (RGA) from the wheat variety Nannong 9918 with powdery mildew resistance characteristic. The amplified product about 540 bp were cloned and sequenced. The sequences belong to three different contigs, and contain the conserved motifs of NB-ARC such as P-loop, kinase-2a, kinase-3a and HD. Phylogenetic analysis of deduced amino acid sequences showed that contig2 was homologous to RPS2 gene (XR138584). The contig2 expression can be induced after powdery mildew inoculation. The sequence distance analysis of deduced amino acid sequences showed contig2 was resistance gene analog (RGA), named as TaRGA. Then, the specific primers were designed based on the sequence of the. resistance gene homologues TaRGA, the full-length cDNA of wheat resistance-related gene TaRGA was obtained through the 5’-RACE and 3’-RACE methods. The cDNA of TaRGA was 3579 bp long including a 2760 bp complete open reading frame (ORF) encoding 919 amino acids. The deduced amino acids of TaRGA protein consisted of a nucleotide binding site (NBS) domain, a leucine-rich repeats (LRR) domain, which were the conserved domains of plant resistance genes. The gene has been submitted to the GenBank datebase, with the accession number of KF725625. Genomics analysis showed that TaRGA contain 2 introns, one intron (319 bp) in TaRGA was located in the region corresponding to the 5’-UTR of mRNA, and the other intron (857 bp) located in the coding domain sequence (CDS) region. Expression analysis via semi-quantitative RT-PCR demonstrated that TaRGA was expressed in the root, leaf, stem and spike tissues, and which was highly expressed in leaf. The TaRGA gene can also be induced by powdery mildew. In addition, the TaRGA expression which was positively regulated by salicylic acid (SA) was detected by real-time RT-PCR.2. Functional analysis of TaRGA by virus-mediated gene silencing and single-cell transinfection assaysThe candidate TaRGA gene was carried out for further functional analysis using BSMV VIGS technique and single-cell transient expression assay. The TaRGA silenced powdery mildew-resistant plants resulted in increased susceptibility to Bgt, and at the infection site, the propagation of conidia was prompted. However, when the TaRGA was expressed in leaf segments by single-cell transient expression assay, the TaRGA-overexpressing leaves showed highly increased defense responses to Bgt, such as enhanced the callose deposition and phenolic autofluorogen accumulation at the pathogen-host interaction sites. Meanwhile, the expression of pathogenesis-related genes decreased in the TaRGA-silenced plants and increased in the TaRGA-transient overexpressing leaf segments. Thus, the above results implied that the TaRGA gene positively participated in the defense responses to powdery mildew disease in wheat.3. Transgenic expression of Hpal10-42 enhances resistance to powdery mildew and Fusarium head blightHpal 10-42, a fragment comprising residues 10 through 42, from the N-terminal region of harpin protein Hpal produced by a bacterial pathogen of rice promotes plant growth and induces disease resistance, and in both effects the Hpal 1042 is 1.3-7.5-fold more effective than the full length. This study evaluated the transgenic expression of the Hpalw-42 gene for resistance to powdery mildew and Fusarium head blight in wheat. The transformation vector pAHC25-Hpal10-42 andpCAMB1A1300-Hpal10-42 were correctly constructed, which expressed highly in monocot plants. After relative molecular analyze, we found that Hpal10-42 gene was integrated into the transgenic lines, inherited from To to T3, and expressed differently in the wheat background. Following inoculation with the B. graminis f. sp. tritici isolate Bgtl5 in the greenhouse, five transgenic lines had significantly higher levels of resistance to powdery mildew compared with nontransformed Yangmai 16. This study also shows that transgenic expression of Hpal10-42 in wheat reduces the severity of Fusarium head blight. Six Hpal10-42-expressing wheat lines were tested and the expression of Hpal10-42 was found to be consistent in T3-T5 progenies. Following inoculation with an indigenous F. graminearum isolate, transgenic lines have significantly lower levels of Fusarium head blight compared to non-transformed plants. Reduction of disease severity is consistent in T3-T5 progenies of Hpal10-42-expressing lines and is equivalent to that in non-transformed plants treated with a fungicide. Therefore, transgenic Hpal10-42 expression may provide an effective measure for management of powdery mildew and Fusarium head blight in the agriculturally significant crop. |
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