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 nucleustargeted transcriptional output sensors and the simultaneous constitutive expression of spectrally separated nuclear reference and plasma membrane-localized reporters. This setup 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 spatiotemporal 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.
Coffee (Coffea spp.) is an economically important crop widely cultivated in (sub) tropical countries worldwide. Commercial coffee production relies mainly on two related species, namely C. arabica and C. canephora. Due to their perennial growth habit, cultivation practices, and narrow genetic diversity, coffees are constantly exposed to many diseases and pests. Coffee leaf rust (Hemileia vastatrix Berk. et Br.), coffee berry disease (Colletotrichum kahawae Bridge and Waller), and coffee wilt disease (Gibberella xylarioides Heim and Saccas/Fusarium xylarioides) are the top fungal diseases affecting C. arabica and C. canephora production areas worldwide. In many regions, chemical-based control measures are widely used and are the only way to control the diseases. Developing resistant cultivars is one of the prerequisites for increasing sustainable market demand and agriculture. However, desired and required resistance traits are not always available in the gene pool. Furthermore, from other crops it is clear that dominant resistance genes introduced into varieties are not durable because of pathogen variability and the emergence of new races of the different pathogens. Utilization of altered susceptibility genes (S genes) offers a novel and alternative strategy for the breeding of durable and broad-spectrum resistance. The S gene encodes a host factor that facilitates a compatible interaction with the pathogen, and impairment of S genes leads to loss-of-susceptibility. In this review, guidelines for effective identification, characterization, and utilization of dysfunctional S genes are proposed to aid breeding activities in order to introduce durable resistance in Coffea spp. Several candidate S genes likely contributing to the susceptibility of Colletotrichum spp., Fusarium spp., and Meloidogyne spp. are discussed. With the rapid development of genetic engineering techniques, including CRISPR-associated systems, we now have the potential to accelerate the application of S genes to achieve durable resistance in coffee.
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.
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.
Verticillium dahliae is a soil-borne fungal pathogen that causes vascular wilt disease in numerous plant species. The only described qualitative resistances against V. dahliae are the Ve1 gene and the V2 locus in tomato. These resistances have been overcome by virulent strains. We tried to identify additional resistances. Out of the methods we tested, comparing the canopy area of V. dahliae-inoculated plants with mock-inoculated plants yielded the best discriminative power in resistance tests. Out of six wild tomato accessions that were previously reported to possess some resistance, Solanum pimpinellifolium G1.1596 and S. cheesmanii G1.1615 displayed the lowest stunting and the least colonization by V. dahliae. Recombinant inbred line (RIL) populations were developed of both populations. No QTLs were identified in the G1.1596 RIL population. In the G1.1615 population, four small-effect QTLs were associated with reduced stunting. Many studies in other hosts also failed to discover major resistance genes against V. dahliae. We hypothesize that the scarcity of major resistance genes against V. dahliae is caused by its endophytic behaviour in nature. The limited damage in nature would not lead to evolutionary pressure to evolve major resistances. However, in agriculture V. dahliae can behave more pathogenic, leading to serious damage.
Verticillium dahliae is a soil‐borne fungal pathogen that causes vascular wilt disease in numerous plant species. The only described qualitative resistances against V. dahliae are the Ve1 gene and the V2 locus in tomato. These resistances have been overcome by virulent strains. We tried to identify additional resistances. Out of the methods we tested, comparing the canopy area of V. dahliae‐inoculated plants with mock‐inoculated plants yielded the best discriminative power in resistance tests. Out of six wild tomato accessions that were previously reported to possess some resistance, Solanum pimpinellifolium G1.1596 and Solanum cheesmanii G1.1615 displayed the lowest stunting and the least colonization by V. dahliae. Recombinant inbred line (RIL) populations were developed of both populations. No QTLs were identified in the G1.1596 RIL population. In the G1.1615 population, four small‐effect QTLs were associated with reduced stunting. Many studies in other hosts also failed to discover major resistance genes against V. dahliae. We hypothesize that the scarcity of major resistance genes against V. dahliae is caused by its endophytic behaviour in nature. The limited damage in nature would not lead to evolutionary pressure to evolve major resistances. However, in agriculture, V. dahliae can behave more pathogenic, leading to serious damage.
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