Constitutive Pathogenesis-related Gene Expression Research Articles

RIN13-mediated disease resistance depends on the SNC1-EDS1/PAD4 signaling pathway in Arabidopsis.

Plants have evolved an innate immune system to protect themselves from pathogen invasion with the help of intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, though the mechanisms remain largely undefined. RIN13 (RPM1-interacting protein 13) was previously reported to enhance disease resistance, and suppress RPM1 (a CNL-type NLR)-mediated hypersensitive response in Arabidopsis via an as yet unknown mechanism. Here, we show that RIN13 is a nuclear-localized protein, and functions therein. Overexpression of RIN13 leads to autoimmunity with high accumulation of salicylic acid (SA), constitutive expression of pathogenesis-related genes, enhanced resistance to a virulent pathogen, and dwarfism. In addition, genetic and transcriptome analyses show that SA-dependent and SA-independent pathways are both required for RIN13-mediated disease resistance, with the EDS1/PAD4 complex as an integration point. RIN13-induced dwarfism was rescued completely by either the pad4-1 or the eds1-2 mutant but partially by snc1-r1, a mutant of the TNL gene SNC1, suggesting the involvement of EDS1/PAD4 and SNC1 in RIN13 functioning. Furthermore, transient expression assays indicated that RIN13 promotes the nuclear accumulation of PAD4. Collectively, our study uncovered a signaling pathway whereby SNC1 and EDS1/PAD4 act together to modulate RIN13-triggered plant defense responses.

5-Aminolevulinic Acid Dehydratase Gene Dosage Affects Programmed Cell Death and Immunity.

Programmed cell death (PCD) is an important form to protect plants from pathogen attack. However, plants must precisely control the PCD process under microbe attacks to avoid detrimental effects. The complexity of how plants balance the defense activation and PCD requires further clarification. Lesion mimic mutants constitute an excellent material to study the crosstalk between them. Here, we identified a Gossypium hirsutum (cotton) lesion mimic mutant (Ghlmm), which exhibits necrotic leaf damage and enhanced disease resistance. Map-based cloning demonstrated that GhLMMD, encoding 5-aminolevulinic acid dehydratase and located on chromosome D5, was responsible for the phenotype. The mutant was resulted from a nonsense mutation within the coding region of GhLMMD It exhibited an overaccumulation of the 5-aminolevulinic acid, elevated levels of reactive oxygen species and salicylic acid, along with constitutive expression of pathogenesis-related genes and enhanced resistance to the Verticillium dahliae infection. Interestingly, GhLMM plays a dosage-dependent role in regulating PCD of cotton leaves and resistance to V. dahliae infection. This study provides a new strategy on the modulation of plant immunity, particularly in polyploidy plants.

A chloroplast-localized protein LESION AND LAMINA BENDING affects defence and growth responses in rice.

Understanding how plants allocate their resources to growth or defence is of long-term importance to the development of new and improved varieties of different crops. Using molecular genetics, plant physiology, hormone analysis and Next-Generation Sequencing (NGS)-based transcript profiling, we have isolated and characterized the rice (Oryza sativa) LESION AND LAMINA BENDING (LLB) gene that encodes a chloroplast-targeted putative leucine carboxyl methyltransferase. Loss of LLB function results in reduced growth and yield, hypersensitive response (HR)-like lesions, accumulation of the antimicrobial compounds momilactones and phytocassanes, and constitutive expression of pathogenesis-related genes. Consistent with these defence-associated responses, llb shows enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae). The lesion and resistance phenotypes are likely to be caused by the over-accumulation of jasmonates (JAs) in the llb mutant including the JA precursor 12-oxo-phytodienoic acid. Additionally, llb shows an increased lamina inclination and enhanced early seedling growth due to elevated brassinosteroid (BR) synthesis and/or signalling. These findings show that LLB functions in the chloroplast to either directly or indirectly repress both JA- and BR-mediated responses, revealing a possible mechanism for controlling how plants allocate resources for defence and growth.

Overexpression of Arabidopsis ACBP3 enhances NPR1-dependent plant resistance to Pseudomonas syringe pv tomato DC3000.

ACBP3 is one of six Arabidopsis (Arabidopsis thaliana) genes, designated ACBP1 to ACBP6, that encode acyl-coenzyme A (CoA)-binding proteins (ACBPs). These ACBPs bind long-chain acyl-CoA esters and phospholipids and are involved in diverse cellular functions, including acyl-CoA homeostasis, development, and stress tolerance. Recombinant ACBP3 binds polyunsaturated acyl-CoA esters and phospholipids in vitro. Here, we show that ACBP3 plays a role in the plant defense response to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. ACBP3 mRNA was up-regulated upon pathogen infection and treatments using pathogen elicitors and defense-related phytohormones. Transgenic Arabidopsis ACBP3 overexpressors (ACBP3-OEs) showed constitutive expression of pathogenesis-related genes (PR1, PR2, and PR5), cell death, and hydrogen peroxide accumulation in leaves. Consequently, ACBP3-OEs displayed enhanced resistance to the bacterial pathogen P. syringae DC3000. In contrast, the acbp3 T-DNA insertional mutant was more susceptible and exhibited lower PR gene transcript levels upon infection. Using the ACBP3 OE-1 line in combination with nonexpressor of PR genes1 (npr1-5) or coronatine-insensitive1 (coi1-2), we concluded that the enhanced PR gene expression and P. syringae DC3000 resistance in the ACBP3-OEs are dependent on the NPR1-mediated, but not the COI1-mediated, signaling pathway. Given that ACBP3-OEs showed greater susceptibility to infection by the necrotrophic fungus Botrytis cinerea while the acbp3 mutant was less susceptible, we suggest that ACBP3 plays a role in the plant defense response against biotrophic pathogens that is distinct from necrotrophic pathogens. ACBP3 function in plant defense was supported further by bioinformatics data showing up-regulation of many biotic and abiotic stress-related genes in ACBP3 OE-1 in comparison with the wild type.

Cyst Nematode Parasitism of Arabidopsis thaliana Is Inhibited by Salicylic Acid (SA) and Elicits Uncoupled SA-Independent Pathogenesis-Related Gene Expression in Roots

Compatible plant-nematode interactions involve the formation of an elaborate feeding site within the host root that requires the evasion of plant defense mechanisms by the parasite. Little is known regarding plant defense signaling pathways that limit nematode parasitism during a compatible interaction. Therefore, we utilized Arabidopsis thaliana mutants perturbed in salicylic acid (SA) biosynthesis or signal transduction to investigate the role of SA in inhibiting parasitism by the beet cyst nematode Heterodera schachtii. We determined that SA-deficient mutants (sid2-1, pad4-1, and NahG) exhibited increased susceptibility to H. schachtii. In contrast, SA-treated wild-type plants showed decreased H. schachtii susceptibility. The npr1-2 and npr1-3 mutants, which are impaired in SA signaling, also showed increased susceptibility to H. schachtii, whereas the npr1-suppressor mutation sni1 showed decreased susceptibility. Constitutive pathogenesis-related (PR) gene-expressing mutants (cpr1 and cpr6) did not show altered susceptibility to H. schachtii; however, constitutive PR gene expression was restricted to cpr1 shoots with wild-type levels of PR-1 transcript present in cpr1 roots. Furthermore, we determined that H. schachtii infection elicits SA-independent PR-2 and PR-5 induction in wild-type roots, while PR-1 transcript and total SA levels remained unaltered. This was in contrast to shoots of infected plants where PR-1 transcript abundance and total SA levels were elevated. We conclude that SA acts via NPR1 to inhibit nematode parasitism which, in turn, is negatively regulated by SNI1. Our results show an inverse correlation between root basal PR-1 expression and plant susceptibility to H. schachtii and suggest that successful cyst nematode parasitism may involve a local suppression of SA signaling in roots.

Analysis of Arabidopsis Growth Factor Gene 1 (GFG1) encoding a nudix hydrolase during oxidative signaling

Maintenance of pyridine nucleotide homeostasis is vital for normal growth and development of plants and animals. We demonstrate that Arabidopsis Growth Factor Gene 1 (GFG1; At4g12720) encoding a nudix hydrolase, is an NADH pyrophosphatase and ADP-ribose pyrophosphatase. The affinity for NADH and ADP-ribose indicates that this enzyme could serve as a connection between sensing cellular redox changes and downstream signaling. GFG1 transcript levels were rapidly and transiently induced during both biotic stresses imposed by avirulent pathogens and abiotic stresses like ozone and osmoticum. T-DNA knock out plants of GFG1 gene, gfg1-1, exhibit pleiotropic phenotypes such as reduced size, increased levels of reactive oxygen species and NADH, microscopic cell death, constitutive expression of pathogenesis-related genes and enhanced resistance to bacterial pathogens. The recombinant protein failed to complement the mutator deficiency in SBMutT- strain of Escherichia coli, suggesting this protein may not play a role in sanitizing the nucleotide pool. Based on rapid transcriptional changes in response to various stresses, substrate specificity of the enzyme, and analysis of the knock out mutant, we propose that GFG1 is a key gene linking cellular metabolism and oxidative signaling.

Overexpression ofNtERF5, a New Member of the Tobacco Ethylene Response Transcription Factor Family Enhances Resistance toTobacco mosaic virus

A new member of the tobacco (Nicotiana tabacum) AP2/ERF (ethylene response factor) transcription factor family, designated NtERF5, has been isolated by yeast one-hybrid screening. In vitro, recombinant NtERF5 protein weakly binds GCC box cis-elements, which mediate pathogen-regulated transcription of several PR (pathogenesis related) genes. NtERF5 transcription is transiently activated by wounding, by infection with the bacterial pathogen Pseudomonas syringae, as well as by inoculation with Tobacco mosaic virus (TMV). In contrast, NtERF5 transcription is not enhanced after application of salicylic acid, jasmonic acid, or ethylene. Constitutive overexpression of NtERF5 (ERF5-Oex) under control of the 35S promoter results in no visible alterations in plant growth or enhanced resistance to Pseudomonas infection. Furthermore, no constitutive expression of PR genes has been observed. In contrast, ERF5-Oex plants show enhanced resistance to TMV with reference to reduced size of local hypersensitive-response lesions and impaired systemic spread of the virus. Since, in TMV-infected ERF5-Oex plants, the viral RNA accumulates only up to 10 to 30% of the wild-type level, we suggest that NtERF5-regulated gene expression is controlling resistance to viral propagation. Previous research has demonstrated that overexpression of ERF genes enhances resistance to bacterial and fungal pathogens. Here, we provide further evidence that resistance to viral infection can be engineered by overexpression of ERF transcription factors.

A Gain-of-Function Mutation in a Plant Disease Resistance Gene Leads to Constitutive Activation of Downstream Signal Transduction Pathways in suppressor of npr1-1, constitutive 1

Plants have evolved sophisticated defense mechanisms against pathogen infections, during which resistance (R) genes play central roles in recognizing pathogens and initiating defense cascades. Most of the cloned R genes share two common domains: the central domain, which encodes a nucleotide binding adaptor shared by APAF-1, certain R proteins, and CED-4 (NB-ARC), plus a C-terminal region that encodes Leu-rich repeats (LRR). In Arabidopsis, a dominant mutant, suppressor of npr1-1, constitutive 1 (snc1), was identified previously that constitutively expresses pathogenesis-related (PR) genes and resistance against both Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2. The snc1 mutation was mapped to the RPP4 cluster. In snc1, one of the TIR-NB-LRR-type R genes contains a point mutation that results in a single amino acid change from Glu to Lys in the region between NB-ARC and LRR. Deletions of this R gene in snc1 reverted the plants to wild-type morphology and completely abolished constitutive PR gene expression and disease resistance. The constitutive activation of the defense responses was not the result of the overexpression of the R gene, because its expression level was not altered in snc1. Our data suggest that the point mutation in snc1 renders the R gene constitutively active without interaction with pathogens. To analyze signal transduction pathways downstream of snc1, epistasis analyses between snc1 and pad4-1 or eds5-3 were performed. Although the resistance signaling in snc1 was fully dependent on PAD4, it was only partially affected by blocking salicylic acid (SA) synthesis, suggesting that snc1 activates both SA-dependent and SA-independent resistance pathways.

Regulation of Arabidopsis COPINE 1 gene expression in response to pathogens and abiotic stimuli.

The copines are a widely distributed class of calcium-dependent, phospholipid-binding proteins of undetermined biological function. Mutation of the Arabidopsis CPN1 (COPINE 1) gene causes a humidity-sensitive lesion mimic phenotype with increased resistance to a bacterial and an oomyceteous pathogen, constitutive pathogenesis-related gene expression, and an accelerated hypersensitive cell death defense response. Here, we show that the disease resistance phenotype of the cpn1-1 mutant was also temperature sensitive, demonstrate increased CPN1 gene transcript accumulation in wild-type plants under low-humidity conditions, and present a detailed analysis of CPN1 gene transcript accumulation in response to bacterial pathogens. In wild-type plants, CPN1 transcript accumulation was rapidly, locally, and transiently induced by both avirulent and virulent strains of Pseudomonas syringae pv tomato bacteria. However, induction of CPN1 transcript accumulation by avirulent bacteria was much faster and stronger than that induced by virulent bacteria. Bacterial induction of CPN1 transcript accumulation was dependent on a functional type III bacterial protein secretion system. In planta expression of the avrRpt2 avirulence gene was sufficient to trigger rapid CPN1 transcript accumulation. CPN1 transcript accumulation was induced by salicylic acid treatment but was not observed during lesion formation in the lesion mimic mutants lsd1 and lsd5. These results are consistent with CPN1 playing a role in plant disease resistance responses, possibly as a suppressor of defense responses including the hypersensitive cell death defense response. The results also suggest that CPN1 may represent a link between plant disease resistance and plant acclimation to low-humidity and low-temperature conditions.

A humidity-sensitive Arabidopsis copine mutant exhibits precocious cell death and increased disease resistance.

The copines are a newly identified class of calcium-dependent, phospholipid binding proteins that are present in a wide range of organisms, including Paramecium, plants, Caenorhabditis elegans, mouse, and human. However, the biological functions of the copines are unknown. Here, we describe a humidity-sensitive copine mutant in Arabidopsis. Under nonpermissive, low-humidity conditions, the cpn1-1 mutant displayed aberrant regulation of cell death that included a lesion mimic phenotype and an accelerated hypersensitive response (HR). However, the HR in cpn1-1 showed no increase in sensitivity to low pathogen titers. Low-humidity-grown cpn1-1 mutants also exhibited morphological abnormalities, increased resistance to virulent strains of Pseudomonas syringae and Peronospora parasitica, and constitutive expression of pathogenesis-related (PR) genes. Growth of cpn1-1 under permissive, high-humidity conditions abolished the increased disease resistance, lesion mimic, and morphological mutant phenotypes but only partially alleviated the accelerated HR and constitutive PR gene expression phenotypes. The disease resistance phenotype of cpn1-1 suggests that the CPN1 gene regulates defense responses. Alternatively, the primary function of CPN1 may be the regulation of plant responses to low humidity, and the effect of the cpn1-1 mutation on disease resistance may be indirect.

Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance.

In many plant-pathogen interactions, resistance is associated with the synthesis and accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. At least two general classes of mutants with altered resistance to pathogen attack have been identified in Arabidopsis. One class exhibits increased susceptibility to pathogen infection; the other class exhibits enhanced resistance to pathogens. In an attempt to identify mutations in resistance-associated loci, we screened a population of T-DNA tagged Arabidopsis thaliana ecotype Wassilewskija (Ws) for mutants showing constitutive expression of the PR-1 gene (cep). A mutant was isolated and shown to constitutively express PR-1, PR-2, and PR-5 genes. This constitutive phenotype segregated as a single recessive trait in the Ws genetic background. The mutant also had elevated levels of SA, which are responsible for the cep phenotype. The cep mutant spontaneously formed hypersensitive response (HR)-like lesions on the leaves and cotyledons and also exhibited enhanced resistance to virulent bacterial and fungal pathogens. Genetic analyses of segregating progeny from outcrosses to other ecotypes unexpectedly revealed that alterations in more than one gene condition the constitutive expression of PR genes in the original mutant. One of the mutations, designated cpr20, maps to the lower arm of chromosome 4 and is required for the cep phenotype. Another mutation, which has been termed cpr21, maps to chromosome 1 and is often, but not always, associated with this phenotype. The recessive nature of the cep trait suggests that the CPR20 and CPR21 proteins may act as negative regulators in the disease resistance signal transduction pathway.

Uncoupling PR Gene Expression from NPR1 and Bacterial Resistance: Characterization of the Dominant Arabidopsis cpr6-1 Mutant

In Arabidopsis, NPR1 mediates the salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance (SAR). Here, we report the identification of another component, CPR 6, that may function with NPR1 in regulating PR gene expression. The dominant CPR 6-1 mutant expresses the SA/NPR1-regulated PR genes (PR-1, BGL 2, and PR-5) and displays enhanced resistance to Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 in the absence of SAR induction. cpr 6-1-induced PR gene expression is not suppressed in the cpr 6-1 npr1-1 double mutant but is suppressed when SA is removed by salicylate hydroxylase. Thus, constitutive PR gene expression in cpr 6-1 requires SA but not NPR1. In addition, resistance to P. s. maculicola ES4326 is suppressed in the cpr 6-1 npr1-1 double mutant, despite expression of PR-1, BGL 2, and PR-5. Resistance to P. s. maculicola ES4326 must therefore be accomplished through unidentified antibacterial gene products that are regulated through NPR1. These results show that CPR 6 is an important regulator of multiple signal transduction pathways involved in plant defense.