SUMMARYArabidopsis downy mildew resistant 6 (dmr6) mutants have lost their susceptibility to the downy mildew Hyaloperonospora arabidopsidis. Here we show that dmr6 is also resistant to the bacterium Pseudomonas syringae and the oomycete Phytophthora capsici. Resistance is accompanied by enhanced defense gene expression and elevated salicylic acid levels. The suppressive effect of the DMR6 oxygenase was confirmed in transgenic Arabidopsis lines overexpressing DMR6 that show enhanced susceptibility to H. arabidopsidis, P. capsici, and P. syringae. Phylogenetic analysis of the superfamily of 2-oxoglutarate Fe(II)-dependent oxygenases revealed a subgroup of DMR6-LIKE OXYGENASEs (DLOs). Within Arabidopsis, DMR6 is most closely related to DLO1 and DLO2. Overexpression of DLO1 and DLO2 in the dmr6 mutant restored the susceptibility to downy mildew indicating that DLOs negatively affect defense, similar to DMR6. DLO1, but not DLO2, is co-expressed with DMR6, showing strong activation during pathogen attack and following salicylic acid treatment. DMR6 and DLO1 differ in their spatial expression pattern in downy mildew-infected Arabidopsis leaves; DMR6 is mostly expressed in cells that are in contact with hyphae and haustoria of H. arabidopsidis, while DLO1 is expressed mainly in the vascular tissues near infection sites. Strikingly, the dmr6-3_dlo1 double mutant, that is completely resistant to H. arabidopsidis, showed a strong growth reduction that was associated with high levels of salicylic acid. We conclude that DMR6 and DLO1 redundantly suppress plant immunity, but also have distinct activities based on their differential localization of expression.
Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.
SummaryThe Arabidopsis mutant downy mildew resistant 6 (dmr6) carries a recessive mutation that results in the loss of susceptibility to Hyaloperonospora parasitica. Here we describe the map-based cloning of DMR6 (At5g24530), which was found to encode a 2-oxoglutarate (2OG)-Fe(II) oxygenase of unknown function. DMR6 transcription is locally induced during infections with both compatible and incompatible H. parasitica isolates. High DMR6 transcript levels were also observed in constitutive defense mutants and after treatment with salicylic acid analog BTH, suggesting that DMR6 has a role during plant defense. Expression analysis of dmr6 mutants, using DNA microarrays and quantitative PCR, showed the enhanced expression of a subset of defense-associated genes, including DMR6 itself, suggesting dmr6-mediated resistance results from the activation of plant defense responses. Alternatively, resistance could be caused by the accumulation of a toxic DMR6 substrate, or by the absence of a DMR6 metabolic product that is required for H. parasitica infection.
Plant disease resistance is commonly triggered by early pathogen recognition and activation of immunity. An alternative form of resistance is mediated by recessive downy mildew resistant 1 (dmr1) alleles in Arabidopsis thaliana. Map-based cloning revealed that DMR1 encodes homoserine kinase (HSK). Six independent dmr1 mutants each carry a different amino acid substitution in the HSK protein. Amino acid analysis revealed that dmr1 mutants contain high levels of homoserine that is undetectable in wild-type plants. Surprisingly, the level of amino acids downstream in the aspartate (Asp) pathway was not reduced in dmr1 mutants. Exogenous homoserine does not directly affect pathogen growth but induces resistance when infiltrated in Arabidopsis. We provide evidence that homoserine accumulation in the chloroplast triggers a novel form of downy mildew resistance that is independent of known immune responses.
The phytohormone jasmonic acid (JA) is vital in plant defense and development. Although biosynthesis of JA and activation of JAresponsive gene expression by the bioactive form JA-isoleucine have been well-studied, knowledge on JA metabolism is incomplete. In particular, the enzyme that hydroxylates JA to 12-OH-JA, an inactive form of JA that accumulates after wounding and pathogen attack, is unknown. Here, we report the identification of four paralogous 2-oxoglutarate/Fe(II)-dependent oxygenases in Arabidopsis thaliana as JA hydroxylases and show that they down-regulate JA-dependent responses. Because they are induced by JA we named them JASMONATE-INDUCED OXYGENASES (JOXs). Concurrent mutation of the four genes in a quadruple Arabidopsis mutant resulted in increased defense gene expression and increased resistance to the necrotrophic fungus Botrytis cinerea and the caterpillar Mamestra brassicae. In addition, root and shoot growth of the plants was inhibited. Metabolite analysis of leaves showed that loss of function of the four JOX enzymes resulted in overaccumulation of JA and in reduced turnover of JA into 12-OH-JA. Transformation of the quadruple mutant with each JOX gene strongly reduced JA levels, demonstrating that all four JOXs inactivate JA in plants. The in vitro catalysis of 12-OH-JA from JA by recombinant enzyme could be confirmed for three JOXs. The identification of the enzymes responsible for hydroxylation of JA reveals a missing step in JA metabolism, which is important for the inactivation of the hormone and subsequent down-regulation of JA-dependent defenses.jasmonic acid | 2OG oxygenases | 12-OH-JA | plant defense
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