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Induction of xa27 by the avrxa27 gene in rice confers broad-spectrum resistance to xanthomonas oryzae pv. oryzae and enhanced resistance to xanthomonas oryzae pv. oryzicola

A xa27, genome technology, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as non-detection

Active Publication Date: 2010-01-06
TEMASEK LIFE SCIENCES LABORATORY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Indeed, Northern blot analysis detected only the Xa27 allele but not the Xa27 allele

Method used

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  • Induction of xa27 by the avrxa27 gene in rice confers broad-spectrum resistance to xanthomonas oryzae pv. oryzae and enhanced resistance to xanthomonas oryzae pv. oryzicola
  • Induction of xa27 by the avrxa27 gene in rice confers broad-spectrum resistance to xanthomonas oryzae pv. oryzae and enhanced resistance to xanthomonas oryzae pv. oryzicola
  • Induction of xa27 by the avrxa27 gene in rice confers broad-spectrum resistance to xanthomonas oryzae pv. oryzae and enhanced resistance to xanthomonas oryzae pv. oryzicola

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Experimental program
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Effect test

Embodiment 1

[0069] Materials and methods

[0070] Plasmids, bacterial strains and growth conditions

[0071] The plasmids used in this study were cosmid 99-avrXa27-20 (Gu et al., 2005) and pHM1avrXa27, cloning vector pGEM-T-easy (Promega, WI 53711, USA) and plant transformation vector pC1305.1 (CAMBIA, Canberra, Australia) and pCPR1avrXa27 (preparation described herein). The bacterial strains used in this study were Escherichia coli strain DH10b (Carlsbad, CA92008, USA), Agrobacterium tumefaciens strain AGL1 (Lazo et al., 1991), Xanthobacterium oryzae strain PXO99 A , AXO1947, AXO1947(pHM1avrXa27), K202, ZHE173 and CIAT1185, and B. oryzae strains L8(pHM1) and L8(pHM1avrXa27). Escherichia coli was cultured on Luria-Bertani medium at 37 °C; Agrobacterium tumefaciens strains were cultured on YEB medium at 28 °C; rice bacterial blight strains and rice bacterial spot bacterial strains were cultured on PSA medium at 28 °C (Gu et al. , 2004). The plasmids were introduced into E. coli, A. tum...

Embodiment 2

[0085] Construction of pCPR1avrXa27

[0086] pCPR1avrXa27 was constructed based on CAMBIA vector pC1305.1. A 3482-bp genomic clone of the avrXa27 gene (including the 3411-bp full-length coding sequence (SEQ ID NO: 5)) from the cosmid 99-avrXa27-20 (Gu et al., 2005) was subcloned into pGEM-T-easy Vector (Promega), the intermediate construct pTavrXa27 was generated. The 3234-bp BamHI fragment of the avrXa27 gene in pTavrXa27 was replaced with the BamHI fragment of the avrXa27F2H gene from pZWavrXa27 (Gu et al., 2005), generating pTZWavrXa27. The SacII-AseI fragment from pTZWavrXa27 (containing the avrXa27 gene (SEQ ID NO: 1)) was isolated, blunt-ended, and cloned into the rice PR1 promoter in pC1305.1 (Gu et al., 2005; SEQ ID NO: 5 ) downstream, resulting in pCPR1avrXa27 ( figure 1 ). The primer pair used to determine the presence of the PR1 promoter and the nopaline synthase gene (Tnos) in transgenic plants were PF / PR (5'-CCATGATTACGAATTCGAGCTCGG-'3 (SEQ ID NO: 6) and 5'-AA...

Embodiment 3

[0088] Generation of avrXa27 transgenic plants

[0089] With the binary plasmid pCPR1avrXa27 ( figure 1 ) Agrobacterium-mediated transformation of the Nipponbare variety to generate avrXa27 transgenic plants. pCPR1avrXa27 carries P in its T-DNA region PR1 -αvrXα27-Tnos fusion gene. A total of 34 independent transgenic T 0 plants. However, after molecular analysis, only four strains of T 0 The plants (L1, L14, L24 and L25 lines) carried the complete αvrXα27 gene. Among the four transgenic plants ( figure 2 , lanes 5, 7, 9 and 11), Southern blot analysis detected the region corresponding to the coding region of αvrXα27 ( figure 1 3.2-kb band of predicted BαmHI fragment of ). At L24 ( figure 2 , lane 8), a 4.5-kb band corresponding to the predicted Nde I-XbαI fragment containing the PR1 promoter and avrXa27 coding region ( figure 1 ). However, this 4.5-kb NdeI-XbαI band was not detected in L1, L14, and L25. Instead, they all carry Nde I-XbαI bands with a molecular s...

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Abstract

The present invention generally provides a method to generate broad-spectrum resistance to bacterial blight disease in plants. More specifically, the present invention provides a method to generate broad-spectrum resistance to Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight disease of rice, and enhanced resistance to Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak of rice. Xa27, an inducible bacterial blight R gene in rice, was induced by the cognate avrXa27 gene expressed in host. Rice plants carrying the avrXa27 transgene and wild-type Xa27 gene conferred resistance to incompatible and compatible pathogens, and enhanced resistance to X. oryzae pv. oryzicola strain L8. The Xa27-mediated enhanced resistance to X. oryzae pv. oryzicola was also observed in the interaction between IRBB27 and L8 harboring pHM1avrXa27. This was further verified by the fact that the Xa27 gene in IRBB27 was induced by theavrXa27 gene in bacteria. The method can be used to engineer broad-spectrum resistance of rice to bacterial blight and enhanced resistance to bacterial leaf streak. Slight modification of this technique can be applied to control bacterial diseases in other crops.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application 60 / 897,864, filed January 29, 2007. Each application is incorporated herein by reference. Background of the invention [0003] In general, the present invention relates to plant molecular biology and genetic methods for engineering plants for enhanced and broad-spectrum resistance to bacterial diseases. [0004] The publications and other materials used herein to illustrate the background of the invention, particularly the examples that provide additional details of implementation, are incorporated herein by reference, for convenience, hereinafter cited by author and date, and in the accompanying bibliography by author in alphabetical order. [0005] Rice (Oryza sativa) bacterial blight caused by Xanthomonas oryzae pv. oryzae is one of the most destructive bacterial diseases in the world (Mew, 1987). Xanthobacterium oryzae enters the susceptible cul...

Claims

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Application Information

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IPC IPC(8): A01H5/00
CPCC12N15/8281
Inventor 尹中朝田冬生
Owner TEMASEK LIFE SCIENCES LABORATORY
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