Manipulating broad-spectrum disease resistance by suppressing pathogen-induced auxin accumulation in rice

Abstract

Breeding crops with the quality of broad-spectrum disease resistance using genetic resources is one of the principal goals of crop improvement. However, the molecular mechanism of broad-spectrum resistance remains largely unknown. Here, we show that GH3-2, encoding an indole-3-acetic acid (IAA)-amido synthetase, mediates a broad-spectrum resistance to bacterial Xanthomonas oryzae pv oryzae and Xanthomonas oryzae pv oryzicola and fungal Magnaporthe grisea in rice (Oryza sativa). IAA, the major form of auxin in rice, results in rice more vulnerable to the invasion of different types of pathogens, which is at least partly due to IAA-induced loosening of the cell wall, the natural protective barrier of plant cells to invaders. X. oryzae pv oryzae, X. oryzae pv oryzicola, and M. grisea secrete IAA, which, in turn, may induce rice to synthesize its own IAA at the infection site. IAA induces the production of expansins, the cell wall-loosening proteins, and makes rice vulnerable to pathogens. GH3-2 is likely contributing to a minor quantitative trait locus for broad-spectrum resistance. Activation of GH3-2 inactivates IAA by catalyzing the formation of an IAA-amino acid conjugate, which results in the suppression of expansin genes. Thus, GH3-2 mediates basal resistance by suppressing pathogen-induced IAA accumulation. It is expected that, regulated by a pathogen-induced strong promoter, GH3-2 alone may be used for breeding rice with a broad-spectrum disease resistance.

Figure 1.

Colocalization of GH3-2 and disease resistance QTLs. LOD, Logarithm of odds.

Figure 2.

GH3-2 expression was responsive to Xoo infection in RILs and their parents, Zhenshan 97 and Minghui 63. Plants were infected with Xoo strain JL691 at the six-leaf stage. ck, Before infection. Bars represent means (three replicates) ± sd. The “a” or “b” indicates that a significant difference was detected between noninfected and Xoo-infected plants of the same rice line at P < 0.01 or P < 0.05, respectively. Two asterisks or one asterisk indicate that a significant difference was detected between susceptible Zhenshan 97 and other rice lines of the same treatment at P < 0.01 or P < 0.05, respectively.

Figure 3.

GH3-2 expression was responsive to pathogen infection. Bars represent means (three replicates) ± sd. A, GH3-2 expression was induced by infection with M. grisea isolate 9-17-2 in RILs and their parents, Zhenshan 97 and Minghui 63, at the four- to five-leaf stage. The “a” or “b” indicates that a significant difference was detected between noninfected and pathogen-infected plants of the same rice line at P < 0.01 or P < 0.05, respectively. Two asterisks or one asterisk indicate that a significant difference was detected between susceptible Zhenshan 97 and other rice lines of the same treatment at P < 0.01 or P < 0.05, respectively. B, GH3-2 expression was influenced by infection with Xoc strain RH3. ck, Before infection.

Figure 4.

Enhanced resistance of GH3-2-overexpressing T1 plants (D176UM) to Xoo strain PXO61 was associated with increased GH3-2 expression. The “a” indicates a significant difference (P < 0.01) from the wild type (WT). Data represent means (five replicates) ± sd.

Figure 5.

Enzyme activity of GH3-2 in vitro. UFLC-MS/MS analysis of amino acid conjugates of IAA synthesized by recombinant GH3-2 protein in different time courses. ck, Proteins from Escherichia coli transferred with the null vector pET28a. cps, Counts per second. [See online article for color version of this figure.]

Figure 6.

Hormone concentrations in wild-type Mudanjiang 8 and GH3-2-overexpressing lines D176UM11 and D176UM34. Bars represent means (three replicates) ± sd. ck, Before infection; FW, fresh weight. A, Concentrations of IAA, IAA-Asp, JA, and SA after inoculation of Xoo strain PXO61 at the booting stage. B, Concentrations of IAA and IAA-Asp after inoculation of Xoc strain RH3 at the booting stage and inoculation of M. grisea isolate CHL358 at the four-leaf stage.

Figure 7.

Expression of expansin (EXP) genes in GH3-2-overexpressing lines D176UM11 and D176UM34. Plants were inoculated with Xoo strain PXO61 or Xoc strain RH3 at the booting stage or inoculated with M. grisea isolate CHL358 at the four-leaf stage. Bars represent means (three replicates) ± sd. ck, Before infection.

Figure 8.

Exogenous application of IAA increased susceptibility to Xoc strain RH3 and M. grisea isolate CHL358 in rice varieties Mudanjiang 8 and Zhonghua 11. Bars represent means (12 replicates for Xoc infection and 20 replicates for M. grisea infection) ± sd. ck, Before infection.

Figure 9.

Expression of putative IAA synthesis-related genes AAO3 and NIT1 in different rice varieties after infection with Xoo strain PXO61, Xoc strain RH3, or M. grisea isolate CHL358. Bars represent means (three replicates) ± sd. Two asterisks or one asterisk indicate that a significant difference was detected between susceptible and resistant rice lines of the same treatment at P < 0.01 or P < 0.05, respectively. ck, Before infection.

Figure 10.

IAA influenced the expression of AAO3, NIT1, and GH3-2. Bars represent means (three replicates) ± sd. ck, Before IAA treatment.