Abstract

Beet cyst nematode (Heterodera schachtii Schm.) is one of the most damaging pests in sugar beet growing areas around the world. The Hs1pro-1 and cZR3 genes confer resistance to the beet cyst nematode, and both were cloned from sugar beet translocation line (A906001). The translocation line carried the locus from B. procumbens chromosome 1 including Hs1pro-1 gene and resistance gene analogs (RGA), which confer resistance to Heterodera schachtii. In this research, BvHs1pro-1 and BvcZR3 genes were transferred into oilseed rape to obtain different transgenic lines by A. tumefaciens mediated transformation method. The cZR3Hs1pro-1 gene was pyramided into the same plants by crossing homozygous cZR3 and Hs1pro-1 plants to identify the function and interaction of cZR3 and Hs1pro-1 genes. In vitro and in vivo cyst nematode resistance tests showed that cZR3 and Hs1pro-1 plants could be infested by beet cyst nematode (BCN) juveniles, however a large fraction of penetrated nematode juveniles was not able to develop normally and stagnated in roots of transgenic plants, consequently resulting in a significant reduction in the number of developed nematode females. A higher efficiency in inhibition of nematode females was observed in plants expressing pyramiding genes than in those only expressing a single gene. Molecular analysis demonstrated that BvHs1pro-1 and BvcZR3 gene expressions in oilseed rape constitutively activated transcription of plant-defense related genes such as NPR1 (non-expresser of PR1), SGT1b (enhanced disease resistance 1) and RAR1 (suppressor of the G2 allele of skp1). Transcript of NPR1 gene in transgenic cZR3 and Hs1pro-1 plants were slightly up-regulated, while its expression was considerably enhanced in cZR3Hs1pro-1 gene pyramiding plants. The expression of EDS1 gene did not change significantly among transgenic cZR3, Hs1pro-1 and cZR3Hs1pro-1 gene pyramiding plants and wild type. The expression of SGT1b gene was slightly up-regulated in transgenic cZR3 and Hs1pro-1 plants compared with the wild type, however, its expression was not changed in cZR3Hs1pro-1 gene pyramiding plant and had no interaction effect. RAR1 gene expression was significantly up-regulated in transgenic cZR3 and cZR3Hs1pro-1 genes pyramiding plants, but almost no expression was found in Hs1pro-1 transgenic plants. These results show that nematode resistance genes from sugar beet were functional in oilseed rape and conferred BCN resistance by activation of a CC-NBS-LRR R gene mediated resistance response. The gene pyramiding had enhanced resistance, thus offering a novel approach for the BCN control by preventing the propagation of BCN in oilseed rape. The transgenic oilseed rape could be used as a trap crop to offer an alternative method for beet cyst nematode control.

Highlights

  • Beet cyst nematode (Heterodera schachtii Schm.) is an important pest of sugar beet that can cause significant reductions in yield

  • Hs1pro-1 gene is the first beet cyst nematode resistance gene cloned from sugar beet translocation line (A906001) by a map-based cloning strategy [11]

  • We hypothesized that the cZR3 gene may interact with the Hs1pro-1 gene to confer resistance against sugar beet nematode in oilseed rape

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Summary

Introduction

Beet cyst nematode (Heterodera schachtii Schm.) is an important pest of sugar beet that can cause significant reductions in yield. Hs1pro-1 gene is the first beet cyst nematode resistance gene cloned from sugar beet translocation line (A906001) by a map-based cloning strategy [11]. Hunger et al [17] cloned 47 resistance gene analogs (RGAs) from genomic DNA of sugar beet. Because no complete resistance could be observed so far by transgenic sugar beet plants (Cai unpublished data), it is proposed that additional genes are required to confer full resistance [30]. We hypothesized that the cZR3 gene may interact with the Hs1pro-1 gene to confer resistance against sugar beet nematode in oilseed rape. We transferred the Hs1pro-1 and cZR3 genes into oilseed rape using hypocotyl explants by A. tumefaciens mediated transformation method and pyramided the cZR3Hs1pro-1 genes by crossing homozygous transgenic cZR3 and Hs1 plants. We transferred the Hs1pro-1 and cZR3 genes into oilseed rape using hypocotyl explants by A. tumefaciens. LB, T-DNA left border; RB, right border; P35S, Cauliflower mosaic virus (CaMV) 35S promoter; NOS, nopaline synthase terminator; MCS, multi clone site including Xhol restriction site; Hs1pro-1, Beta vulgaris Hs1pro-1 gene open read fragment sequence; cZR3, Beta vulgaris resistance gene sequence; GUS, β-glucuronidase report gene; NPTII, neomycin phosphotranferase gene for kanamycin resistance. (C) Callus induction from hypocotyl explants after co-cultivation with A. tumefaciens. (D) Shoots regenerated on SIM. (E) Shoots elongation on SEM. (F) Shoots rooted on RM. (G,H) PCR assay for transgenic Hs1pro-1 plants (G) and cZR3 plants (H) M means 1 kb DNA ladder; P means Hs1 or cZR3 plasmid DNA, W means non-transgenic plant DNA; Hs1-1, Hs1-2 and Hs1-3 mean T0 independent transgenic Hs1pro-1 plants; cZR3-1, cZR3-2 and cZR3-3 mean T0 independent transgenic cZR3 plants. (I) Southern blot assay for T0 independent transgenic cZR3 and Hs1pro-1 lines, P1P2 means Hs1 and cZR3 plasmid DNA, respectively; 1, 3, and 5 mean T0 independent transgenic Hs1 plants; 2, 4, and 6 mean T0 independent transgenic cZR3 plants

Generation of cZR3Hs1pro-1 Gene Pyramiding Plants
Plant Transformation
PCR Analysis
Southern Blot Analysis
Histochemical GUS Assays
Genes Pyramiding and Progeny Analysis
Nematode Resistance Assay In Vitro and In Vivo
Semi Real Time PCR Analysis
Findings
Data Analysis
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