| Rice is the most important staple food all over the world. Bacterial blight (BB), bacterial leaf streak (BLS) and blast fungus are main reasons for the great loss in rice production. Exploiting rice defense-responsive genes with broad-spectrum disease resistance provides theoretic basis for breeding rice varieties of good quality. Here we present the function of GH3-2, one of the rice GH3 gene family members, in rice resistance responses by means of advanced techniques in plant molecular biology and biochemistry fields.We performed QTL analyses of GH3-2 and confirmed that GH3-2 co-localized with disease resistance QTLs against Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe grisea in chromosome 1 respectively. We overexpressed GH3-2 in susceptible Japonica variety Mudanjiang 8 and suppressed GH3-2 in moderately susceptible Japonica variety Zhonghua 11 by Agrobacterium-mediated transformation. GH3-2-overexpressing plants showed dwarf phenotype and increased resistance to Xoo, M. grisea and Xanthomonas oryzae pv. oryzicola (Xoc). According to Northern analysis, the phenotypes of GG3-2-overexpressing transgenic plants were co-segregated with the GH3-2 expression level. However, GH3-2-suppressing plants were not distinguishable from wild type and transgenic negative plants in height and performance to pathogen infection, which could be explained by functional redundancy among the family members. We detected the expression pattern of some defense-responsive genes and expansin genes in transgenic overexpression plants and wild-type plants after Xoo infection by quantitative RT-PCR. The expression levels of pathogenesis-related (PR) genes PR1a and PR1b, salicylic acid (SA) synthesis-related genes PAD4 and PALI, jasmonic acid (JA) synthesis-related genes LOX, AOS2 and PR10, and three expansin genes EXPA1, EXPA5 and EXPA10 were depressed in overexpression plants. The concentration of SA and JA in transgenic overexpression plants also decreased compared with wild-type plants. These results suggest that the resistance against Xoo mediated by GH3-2 may not rely on the activation of the SA-or JA-dependent pathways. It may be related with the suppression of cell expansion and the resulted compact clusters of cells.We expressed and purified the recombinant GH3-2 protein with His tag in vitro. We tested the enzyme activity of GH3-2 and found that GH3-2 had indole-3-acetic acid (IAA)-amido synthetase tactivity. Compared with the wild type plants, the IAA concentration in transgenic overexpression plants was lower; conversely the IAA-Asp concentration was higher, which confirmed GH3-2 to be an IAA-amido synthetase in vivo.Hormone concentration assay showed that IAA level in susceptible wild type Mudanjiang 8 was significantly higher than that in GH3-2 overexpression plants before and after infection of Xoo, Xoc or M. grisea. Two IAA synthesis-related genes NIT1 and AAO3 were up-regulated in plants inoculated with Xoo, M. grisea or Xoc, and relative expression level of these two genes in susceptible variety was higher than that in corresponding resistant variety. These results suggested that the elevated IAA level after pathogen infection was due to up-regulation of IAA synthesis pathway.We pre-treated wild type plants with IAA and then inoculated with Xoc and M. grisea respectively. The result showed that IAA aggravated the disease symptoms of rice against these two pathogens, which indicated that IAA played the role of virulence factor in pathogenesis.We quantified the hormone secreted to liquid culture by Xoo strains PXO61, PXO99 and PXO347, Xoc strain RH3 and M. grisea strains CHL358 and RB11. IAA was found to exist in liquid cultures secretion by these pathogens. This result further demonstrated that pathogens might use IAA as virulence factor during infection process and IAA negatively regulated rice defense response.In conclusion, GH3-2 conferred rice broad-spectrum disease resistance by means of inactivation of IAA and down-regulation of IAA signaling pathway. |
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