Cloning and Analysis of a Disease Resistance Gene Homolog from Soybean

Conserved domains e.g. nucleotide binding site (NBS) were found in several cloned plant disease resistance genes. Based on the NBS domain, resistance gene analogs (RGAs) have been isolated previously and were used as probes to screen a soybean (Glycine max L. Merr.) cDNA library. A full-length cDNA, KR3, was obtained by screening the library and rapid amplification of cDNA ends (RACE) method. Sequence analysis revealed that the cDNA is 2 353 bp in length and the open reading frame (ORF) codes for a polypeptide of 636 amino acids with a Toll-Interleukin-1 receptor (TIR) and a NBS domain. Sequence alignment showed that it was similar to N gene of tobacco. The phylogenetic tree analysis of R proteins with NBS from higher plants was performed. The KR3 gene has low copies in soybean genome and its expression was induced by exogenous salicylic acid (SA).

Analysis of three types of resistance gene analogs in Pm U region from Triticum urartu

Resistance gene analog(RGA) screening of mapped disease-resistant genes not only helps to clone these genes but also helps to develop efficient molecular markers for resistance breeding. The present study focused on the PmU region located on chromosome 7 Au L of Triticum urartu, and recently, a nucleotide binding site(NBS)-encoding gene, Pm60, was cloned from the same chromosome arm. In this research, NBS, protein kinase(PK), and ATP-binding cassette(ABC), the three disease resistance-related gene families, were analyzed within PmU region by using informatics tools, and an expression experiment was conducted to verify their functions in vivo. Comparative genomic analysis revealed that 126 RGAs were included on chromosome 7 Au L, and 30 of the RGAs as well as Pm60 were found in the Pm U region. Transcriptome database analysis of T. urartu revealed 14 PmU-RGAs with expression data, and three PmU-NBSs exhibited significant changes in expression after inoculation with Blumeria graminis f. sp. tritici(Bgt); TRIUR314879 was up-regulated, while TRIUR300450 and TRIUR306270 were down-regulated. Cluster analysis showed that these three PmU-NBSs were clustered far from the cloned wheat resistance genes. Then, qRT-PCR was performed to investigate the expression of 14 PmU-RGAs and Pm60 after inoculation with Bgt race E09; the results showed that Pm60 was specifically expressed in UR206 which carrying PmU, but not in susceptible UR203; while TRIUR314879 was significantly up-regulated and TRIUR300450 was downregulated in UR206 after inoculation. These results indicated that PmU is Pm60, and TRIUR314879 and TRIUR300450 may also be involved in the defense against Bgt.

Cloning and Characterization of Full Length cDNA of a CC-NBS-LRR Resistance Gene in Sweetpotato

Conserved domain such as nucleotide binding site （NBS） was found in several cloned plant disease resistance genes. Based on the NBS domain, resistance gene analogues （RGAs） have been isolated. A full-length cDNA, SPR1 was obtained by rapid amplification of cDNA ends （RACE） method. Sequence analysis indicated that the length of SPR1 was 3 066 bp, including a complete open reading frame of 2 667 bp encoding SPR1 protein of 888 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has a typical structure of nonT1R-NBS-LRR genes, with NB-ARC, CC, and LRR domains. The SPR1-related sequences belonged to multicopy gene family in sweetpotato genome according to the result of Southern blotting. Semi-quantitative RT-PCR analysis showed SPR1 expressed in all tested tissues. The cloning of putative resistance gene from sweetpotato provides a basis for studying the structure and function of sweetpotato disease-resistance relating genes and disease resistant genetic breeding in sweetpotato. The gene has been submitted to the GenBank database, and the accession number is EF428453.

Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection

Verticillium wilt of potato is caused by the fungus pathogen Verticillium dahliae. Present sRNA sequencing data revealed that miR482 was in response to V. dahliae infection, but the function in potato is elusive. Here, we characterized potato miR482 family and its putative role resistance to Verticillium wilt. Members of the potato miR482 superfamily are variable in sequence, but all variants target a class of disease-resistance proteins with nucleotide binding site （NBS） and leucine-rich repeat （LRR） motifs. When potato plantlets were infected with V. dahliae, the expression level of miR482e was downregulated, and that of several NBS-LRR targets of miR482e were upregulated. Transgenic potato plantlets overexpressing miR482e showed hypersensitivity to V. dahliae infection. Using sRNA and degradome datasets, we validated that miR482e targets mRNAs of NBS-LRR disease-resistance proteins and triggers the production of trans-acting （ta）- siRNAs, most of which target mRNAs of defense-relatedproteins. Thus, the hypersensitivity of transgenic potato could be explained by enhanced miR482e and miR482e-derived ta- siRNA-mediated silencing on NBS-LRR-disease-resistance pro- teins. It is speculated that a miR482-mediated silencing cascade mechanism is involved in regulating potato resistance against V. dahliae infection and could be a counter defense action of plant in response to pathogen infection.