Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression

Abstract

Although ethylene regulates a wide range of defense-related genes, its role in plant defense varies greatly among different plant-microbe interactions. We compared ethylene's role in plant response to virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria in tomato (Lycopersicon esculentum Mill.). The ethylene-insensitive Never ripe (Nr) mutant displays increased tolerance to the virulent strain, while maintaining resistance to the avirulent strain. Expression of the ethylene receptor genes NR and LeETR4 was induced by infection with both virulent and avirulent strains; however, the induction of LeETR4 expression by the avirulent strain was blocked in the Nr mutant. To determine whether ethylene receptor levels affect symptom development, transgenic plants overexpressing a wild-type NR cDNA were infected with virulent X. campestris pv. vesicatoria. Like the Nr mutant, the NR overexpressors displayed greatly reduced necrosis in response to this pathogen. NR overexpression also reduced ethylene sensitivity in seedlings and mature plants, indicating that, like LeETR4, this receptor is a negative regulator of ethylene response. Therefore, pathogen-induced increases in ethylene receptors may limit the spread of necrosis by reducing ethylene sensitivity.

Figure 1

Disease severity in tomato leaves 14 DAI with X. campestris pv. vesicatoria. A, Four-week-old wild-type (WT) and ethylene-insensitive Nr mutant plants were inoculated with virulent and avirulent strains of the pathogen. B, Four-week-old wild-type and NROE-1 and NROE-2 were infected with a virulent strain of the pathogen.

Figure 2

Growth of X. campestris pv. vesicatoria in leaves of wild-type and Nr tomato plants. Four-week-old plants were inoculated with virulent and avirulent strains of the pathogen. se bars are smaller than the symbols. ○, Wild-type virulent; ●, wild-type avirulent; □, Nr virulent; ▪, Nr avirulent.

Figure 3

Ethylene synthesis in leaves of wild-type and Nr mutant plants inoculated with X. campestris pv. vesicatoria. Four-week-old plants were inoculated with virulent and avirulent strains of the pathogen. □, Control; ░⃞, virulent; ▪, avirulent.

Figure 4

Pathogenesis-related and ethylene biosynthesis gene expression in leaves of wild-type and Nr mutant tomato plants inoculated with X. campestris pv. vesicatoria. Four-week-old plants were inoculated with virulent and avirulent strains of the pathogen. RNA levels were determined by RNA gel-blot analysis. Plants are wild type unless otherwise indicated. C, Control; V, virulent; A, avirulent.

Figure 5

Expression of the tomato ethylene receptor genes LeETR4 and NR in leaves following inoculation with X. campestris pv. vesicatoria. Four-week-old plants were inoculated with virulent and avirulent strains of the pathogen. Percent mRNA was quantified by RNase protection assays as described in “Materials and Methods.” ▪, Control; ▴, virulent; ●, avirulent.

Figure 6

Ethylene receptor gene expression in tomato leaves in response to exogenous ethylene. Four-week-old wild-type (cv Pearson) plants were treated with ethylene for 1 h. Percent mRNA of ethylene receptor genes was quantified by RNase protection assays as described in “Materials and Methods.”

Figure 7

Expression of the ethylene receptor gene NR in stems (black bars), etiolated seedlings (white bars), and leaves (gray bars) of wild-type (WT) and NROE-1 and NROE-2 tomato plants. Percent mRNA was quantified by RNase protection assays as described in “Materials and Methods.”

Figure 8

Triple response assay of NROE-1 and NROE-2, Nr mutant, and wild-type tomato seedlings. Seedling length is the sum of hypocotyl and root length. Seedlings were grown in the dark for 2 weeks on 1% (w/v) agar containing varying concentrations of ACC. Seedlings in the top panel are cv Floradade (●, NROE-1; ▴, NROE-2; □, wild type); seedlings in the bottom panel are cv Pearson (●, Nr; □, wild type).

Figure 9

Electrolyte leakage from tomato leaves infected with a virulent strain of X. campestris pv. vesicatoria. Plants were 4 weeks old at the time of inoculation. ▪, Wild-type control; ●, wild-type virulent; ▴, NROE-1 virulent; ♦, NROE-2 virulent.