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A CC-NBS-LRR type gene GHNTR1 confers resistance to southern root-knot nematode in Nicotiana.benthamiana and Nicotiana.tabacum

Abstract

Root-knot nematodes are obligate parasites that invade the roots of agricultural plants and induce the formation of specialized feeding structures, especially races 3 and 4 of the southern root-knot nematode. However, not much is known about the defense mechanisms of plants against the invasion of M. incognita race 1. In this study, we characterized and performed functional analysis of the CC-NBS-LRR domain gene, GHNTR1. Using the GHNTR1 promoter to drive the GUS marker gene, we found that GUS expression was high in the roots and shoots of seedlings, four leaves stages, and mature stages. When GHNTR1 was transiently expressed in Nicotiana. benthamiana and Nicotiana. tabacum, necrosis was observed in the leaves and detectable amounts of H2O2 had accumulated when compared to the control plants. Stable transformation of N. benthamiana with the GHNTR1 gene using Agrobacterium induced the expression of defense marker genes PR1, PR2, LOX, and ERF1. In addition, the transgenic N. benthamiana and N. tabacum plants exhibited higher resistance to M. incognita infection. When transgenic N. tabacum were challenged with M. incognita, callose deposition and peroxide accumulation were observed in the roots by aniline-blue and DAB staining, respectively. Sequencing and bioinformatic analysis of small RNA revealed 48 miRNAs were up-regulated and 32 miRNAs were down-regulated. Further, 345 genes were predicted as targets of miRNAs and these included genes that are involved in cell death, death, and stress response. Thus, GHNTR1 is the first gene isolated from cotton that confers resistance against M. incognita and it induced a series of hypersensitive responses in transgenic tobacco. These findings improve our understanding of the molecular mechanisms of the response of upland cotton to M. incognita infection.

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Acknowledgments

This work was supported to B.Z. by grants from the National Natural Science Foundation of China (No. 31371930), National Science and Technology Major Project for Transgenic Breeding (No. 2014ZX0800501B) and the fund for Independent Innovation of Agricultural Sciences in Jiangsu Province (No. cx(13) 2029).

Authors’ contributions

BLZ, YWY, JYW and WHZ performed the data analysis and drafted the manuscript. BLZ, YWY, and JYW participated in the analysis of the data. LXT, ZZH, TLL and TZC performed the experiments. All authors approved the final version of the manuscript.

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The authors declare that they have no conflict of interest.

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Correspondence to Wenhua Zhang.

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Supplementary Figure 1
figure6

Relative expression level of GHNTR1 gene in transgenic plants. A. The transgenic N. tabacum plants. B. Transgenic N. benthamiana plants. (JPEG 1136 kb)

Supplementary Figure 2
figure7

The phenotype and expression of PR genes in transgenic N. tabacum plants. A. The phenotype of aboveground parts in transgenic N. tabacum plants. B. Expression level of PR genes without M. incognita infection in transgenic N. tabacum plants. (JPEG 4966 kb)

Supplementary table 1

List of primers used in this study. (XLS 26 kb)

Supplementary table 2

Summary of the predicted miRNA in wild-type and transgenic N. benthamiana plants. (XLS 177 kb)

Supplementary table 3

Annotation of differentially expressed miRNA target genes. (XLS 736 kb)

Supplementary table 4

GO enrichment analysis of the target genes. (XLS 32 kb)

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Zhang, B., Yang, Y., Wang, J. et al. A CC-NBS-LRR type gene GHNTR1 confers resistance to southern root-knot nematode in Nicotiana.benthamiana and Nicotiana.tabacum . Eur J Plant Pathol 142, 715–729 (2015). https://doi.org/10.1007/s10658-015-0646-3

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Keywords

  • Cotton
  • GHNTR1
  • Hypersensitive response
  • Tobacco
  • Small RNA