The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana

Abstract

Majority of plant growth promoting rhizobacteria (PGPR) confer plant immunity against a wide range of foliar diseases by activating plant defences that reduce a plant’s susceptibility to pathogen attack. Here we show that Arabidopsis thaliana (Col-0) plants exposed to Bacillus subtilis strain FB17 (hereafter FB17), results in reduced disease severity against Pseudomonas syringae pv. tomato DC3000 (hereafter DC3000) compared to plants without FB17 treatment. Exogenous application of the B. subtilis derived elicitor, acetoin (3-hydroxy-2-butanone), was found to trigger induced systemic resistance (ISR) and protect plants against DC3000 pathogenesis. Moreover, B. subtilis acetoin biosynthetic mutants that emitted reduced levels of acetoin conferred reduced protection to A. thaliana against pathogen infection. Further analysis using FB17 and defense-compromised mutants of A. thaliana indicated that resistance to DC3000 occurs via NPR1 and requires salicylic acid (SA)/ethylene (ET) whereas jasmonic acid (JA) is not essential. This study provides new insight into the role of rhizo-bacterial volatile components as elicitors of defense responses in plants.

Acknowledgements

H.P.B. acknowledges the support from University of Delaware Research Foundation (UDRF) and NSF Award IOS-0814477. P.W.P. acknowledges the partial financial support from Welch Foundation (Grant D-1478).

Figures and Tables

Figure 1 Pseudomonas syringae DC3000 infection symptom development (A), bacterial multiplication (B), on the plants with or without root inoculation with B. subtilis FB17. The images are a representative sample of n = 6 and the data is an average of two separate experiments with six replicates. The yellowing of leaves in (A) shows classical DC3000 inflicted disease symptoms indicative of chlorosis. Different letters on the bars indicate a statistically significant difference (F_(4,25) = 312.6, p ≤ 0.05, ANOVA).

Figure 2 Effect of acetoin on disease symptom development (A), and pathogen proliferation (B) in the leaves of A. thaliana Col-0 plants leaf inoculated with DC3000. The images are a representative example of n = 6 and the data is an average of two separate experiments each with six replicates. The yellow patches on the leaves in (A), shows classical DC3000 inflicted disease symptoms in the form of chlorosis. Different letters on the bars indicate a statistically significant difference (p < 0.05, t-test).

Figure 3 Effect of root colonization of B. subtilis acetoin biosynthetic mutants (A), on disease symptom development (B) percent disease incidence (C), and pathogen multiplication (D) in the leaves of A. thaliana Col-0 plants inoculated with DC3000. The images are a representative example of n = 6 and the data is an average of two separate experiments each with six replicates. The root confocal images were taken using 10x objective lens. The green fluorescence in (A) shows the FB17 biofilm on the root surface. The yellow patches on the leaves in the (B) show classical DC3000 inflicted disease symptoms in the form of chlorosis (scale bar: A = 100 µm). Different letters on the bars indicate a statistically significant difference between treatments (C: F_(6,43) = 153.1, p < 0.05, ANOVA; D: F_(6,43) = 125.3, p < 0.05, ANOVA).

Figure 4 Defense-compromised A. thaliana plants revealing differential leaf colony titer response of DC3000 under B. subtilis FB17 (A) and acetoin treatment (B). Twenty one days old wild type and disease compromised mutants were root inoculated with FB17 and the leaves were infected with DC3000. The infected plant leaves were used post 96 hr of treatment to enumerate total leaf CFU counts for DC3000. The data is an average of six replicates of two experiments conducted (Mean ± S.D; n = 6). Different letters on the bars indicate a statistically significant difference (F_(2,29) = 231.2, p ≤ 0.05, ANOVA).

Figure 5 (A) Induction of ethylene and JA responsive genes (PDF1.2 & Jin1) 4 days after acetoin challenge. (B) Induction of PR1 and PDF1.2 genes in roots and leaves of FB17 treated plants. Plants were analyzed 4 days after FB17 challenge. Transcript levels were checked in both leaves and roots of mock, FB17 and DC3000 inoculated plants. Panels indicate the transcript levels for two genes (PR1 and PDF1.2) in both leaves and roots of plants. Column labels indicate that plants were mock treated (blunt infiltration in leaves and flooding roots with deionized water), followed by root inoculation with FB17 and infiltration of leaves with DC3000. The data is an average of six replicates of two experiments conducted separately and the images are a representative of six replicates.