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Overexpression of F-Box Nictaba Promotes Defense and Anthocyanin Accumulation in Arabidopsis thaliana After Pseudomonas syringae Infection.

ABSTRACT: Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a well-known pathogen and model organism used to study plant-pathogen interactions and subsequent plant immune responses. Numerous studies have demonstrated the effect of Pst DC3000 on Arabidopsis plants and how type III effectors are required to promote bacterial virulence and pathogenesis. F-Box Nictaba (encoded by At2g02360) is a stress-inducible lectin that is upregulated in Arabidopsis thaliana leaves after Pst DC3000 infection. In this study, a flood inoculation assay was optimized to check the performance of transgenic Arabidopsis seedlings with different expression levels of F-Box Nictaba after bacterial infection. Using a combination of multispectral and fluorescent imaging combined with molecular techniques, disease symptoms, transcript levels for F-Box Nictaba, and disease-related genes were studied in Arabidopsis leaves infected with two virulent strains: Pst DC3000 and its mutant strain, deficient in flagellin ΔfliC. Analyses of plants infected with fluorescently labeled Pst DC3000 allowed us to study the differences in bacterial colonization between plant lines. Overexpression plants showed a reduced bacterial content during the later stages of the infection. Our results show that overexpression of F-Box Nictaba resulted in reduced leaf damage after bacterial infections, whereas knockdown and knockout lines were not more susceptible to Pseudomonas infection than wild-type plants. In contrast to wild-type and knockout plants, overexpressing lines for F-Box Nictaba revealed a significant increase in anthocyanin content, better efficiency of photosystem II (Fv/Fm), and higher chlorophyll content after Pst DC3000 infection. Overexpression of F-Box Nictaba coincided with increased expression of salicylic acid (SA) related defense genes, confirming earlier data that showed that F-Box Nictaba is part of the SA-dependent defense against Pst DC3000 infection. Knockout lines yielded no discernible effects on plant symptoms after Pseudomonas infection suggesting possible gene redundancy between F-Box Nictaba genes.

SUBMITTER: Romero-Perez A

PROVIDER: S-EPMC8358183 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Plant responses to a combination of drought and bacterial pathogen infection, an agronomically important and altogether a new stress, are not well-studied. While occurring concurrently, these two stresses can lead to synergistic or antagonistic effects on plants due to stress-interaction. It is reported that plant responses to the stress combinations consist of both strategies, unique to combined stress and those shared between combined and individual stresses. However, the combined stress response mechanisms governing stress interaction and net impact are largely unknown. In order to study these adaptive strategies, an accurate and convenient methodology is lacking even in model plants like Arabidopsis thaliana. The gradual nature of drought stress imposition protocol poses a hindrance in simultaneously applying pathogen infection under laboratory conditions to achieve combined stress. In present study we aimed to establish systematic combined stress protocol and to study physiological responses of the plants to various degrees of combined stress. Here, we have comprehensively studied the impact of combined drought and Pseudomonas syringae pv. tomato DC3000 infection on A. thaliana. Further, by employing different permutations of drought and pathogen stress intensities, an attempt was made to dissect the contribution of each individual stress effects during their concurrence. We hereby present two main aspects of combined stress viz., stress interaction and net impact of the stress on plants. Mainly, this study established a systematic protocol to assess the impact of combined drought and bacterial pathogen stress. It was observed that as a result of net impact, some physiological responses under combined stress are tailored when compared to the plants exposed to individual stresses. We also infer that plant responses under combined stress in this study are predominantly influenced by the drought stress. Our results show that pathogen multiplication was reduced by drought stress in combined stressed plants. Combined stressed plants also displayed reduced ROS generation and declined cell death which could be attributed to activation of effective basal defense responses. We hypothesize a model on ABA mediated gene regulation to partly explain the possible mechanistic basis for reduced in planta bacterial numbers under combined stress over individual pathogen stress.

Project description:Plant-pathogen interactions involve sophisticated action and counteraction strategies from both parties. Plants can recognize pathogen derived molecules, such as conserved pathogen associated molecular patterns (PAMPs) and effector proteins, and subsequently activate PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. However, pathogens can evade such recognitions and suppress host immunity with effectors, causing effector-triggered susceptibility (ETS). The differences among PTI, ETS, and ETI have not been completely understood. Toward a better understanding of PTI, ETS, and ETI, we systematically examined various defense-related phenotypes of Arabidopsis infected with different Pseudomonas syringae pv. maculicola ES4326 strains, using the virulence strain DG3 to induce ETS, the avirulence strain DG34 that expresses avrRpm1 (recognized by the resistance protein RPM1) to induce ETI, and HrcC(-) that lacks the type three secretion system to activate PTI. We found that plants infected with different strains displayed dynamic differences in the accumulation of the defense signaling molecule salicylic acid, expression of the defense marker gene PR1, cell death formation, and accumulation/localization of the reactive oxygen species, H2O2. The differences between PTI, ETS, and ETI are dependent on the doses of the strains used. These data support the quantitative nature of PTI, ETS, and ETI and they also reveal qualitative differences between PTI, ETS, and ETI. Interestingly, we observed the induction of large cells in the infected leaves, most obviously with HrcC(-) at later infection stages. The enlarged cells have increased DNA content, suggesting a possible activation of endoreplication. Consistent with strong induction of abnormal cell growth by HrcC(-), we found that the PTI elicitor flg22 also activates abnormal cell growth, depending on a functional flg22-receptor FLS2. Thus, our study has revealed a comprehensive picture of dynamic changes of defense phenotypes and cell fate determination during Arabidopsis-P. syringae interactions, contributing to a better understanding of plant defense mechanisms.

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Overexpression of F-Box Nictaba Promotes Defense and Anthocyanin Accumulation in Arabidopsis thaliana After Pseudomonas syringae Infection.

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