Inactivation of tomato WAT1 leads to reduced susceptibility to Clavibacter michiganensis through downregulation of bacterial virulence factors.

Tomato bacterial canker caused by Clavibacter michiganensis (Cm) is considered to be one of the most destructive bacterial diseases of tomato. To date, no resistance to the pathogen has been identified. While several molecular studies have identified (Cm) bacterial factors involved in disease development, the plant genes and mechanisms associated with susceptibility of tomato to the bacterium remain largely unknown. Here, we show for the first time that tomato gene SlWAT1 is a susceptibility gene to Cm. We inactivated the gene SlWAT1 through RNAi and CRISPR/Cas9 to study changes in tomato susceptibility to Cm. Furthermore, we analysed the role of the gene in the molecular interaction with the pathogen. Our findings demonstrate that SlWAT1 functions as an S gene to genetically diverse Cm strains. Inactivation of SlWAT1 reduced free auxin contents and ethylene synthesis in tomato stems and suppressed the expression of specific bacterial virulence factors. However, CRISPR/Cas9 slwat1 mutants exhibited severe growth defects. The observed reduced susceptibility is possibly a result of downregulation of bacterial virulence factors and reduced auxin contents in transgenic plants. This shows that inactivation of an S gene may affect the expression of bacterial virulence factors.

Impairment of Tomato WAT1 Enhances Resistance to Vascular Wilt Fungi Despite Severe Growth Defects.

Verticillium dahliae is a particularly notorious vascular wilt pathogen of tomato and poses a reoccurring challenge to crop protection as limited qualitative resistance is available. Therefore, alternative approaches for crop protection are pursued. One such strategy is the impairment of disease susceptibility (S) genes, which are plant genes targeted by pathogens to promote disease development. In Arabidopsis and cotton, the Walls Are Thin 1 (WAT1) gene has shown to be a S gene for V. dahliae. In this study, we identified the tomato WAT1 homolog Solyc04g080940 (SlWAT1). Transient and stable silencing of SlWAT1, based on virus-induced gene silencing (VIGS) and RNAi, respectively, did not consistently lead to reduced V. dahliae susceptibility in tomato. However, CRISPR-Cas9 tomato mutant lines carrying targeted deletions in SlWAT1 showed significantly enhanced resistance to V. dahliae, and furthermore also to Verticillium albo-atrum and Fusarium oxysporum f. sp. lycopersici (Fol). Thus, disabling the tomato WAT1 gene resulted in broad-spectrum resistance to various vascular pathogens in tomato. Unfortunately these tomato CRISPR mutant lines suffered from severe growth defects. In order to overcome the pleiotropic effect caused by the impairment of the tomato WAT1 gene, future efforts should be devoted to identifying tomato SlWAT1 mutant alleles that do not negatively impact tomato growth and development.

Quantitative Hormone Signaling Output Analyses of Arabidopsis thaliana Interactions With Virulent and Avirulent Hyaloperonospora arabidopsidis Isolates at Single-Cell Resolution.

The phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are central regulators of biotic and abiotic stress responses in Arabidopsis thaliana. Here, we generated modular fluorescent protein-based reporter lines termed COLORFUL-PR1pro, -VSP2pro, and -PDF1.2apro. These feature hormone-controlled nucleus-targeted transcriptional output sensors and the simultaneous constitutive expression of spectrally separated nuclear reference and plasma membrane-localized reporters. This set-up allowed the study of cell-type specific hormone activities, cellular viability and microbial invasion. Moreover, we developed a software-supported high-throughput confocal microscopy imaging protocol for output quantification to resolve the spatio-temporal dynamics of respective hormonal signaling activities at single-cell resolution. Proof-of-principle analyses in A. thaliana leaves revealed distinguished hormone sensitivities in mesophyll, epidermal pavement and stomatal guard cells, suggesting cell type-specific regulatory protein activities. In plant-microbe interaction studies, we found that virulent and avirulent Hyaloperonospora arabidopsidis (Hpa) isolates exhibit different invasion dynamics and induce spatio-temporally distinct hormonal activity signatures. On the cellular level, these hormone-controlled reporter signatures demarcate the nascent sites of Hpa entry and progression, and highlight initiation, transduction and local containment of immune signals.