AtCPK1 calcium‐dependent protein kinase mediates pathogen resistance in Arabidopsis

In mammals, lipid bodies play a key role during pathological and infectious diseases. However, our knowledge on the function of plant lipid bodies, apart from their role as the major site of lipid storage in seed tissues, remains limited. Here, we provide evidence that a calcium‐dependent protein kinase (CPK) mediates pathogen resistance in Arabidopsis. AtCPK1 expression is rapidly induced by fungal elicitors. Loss‐of‐function mutants of AtCPK1 exhibit higher susceptibility to pathogen infection compared to wild‐type plants. Conversely, over‐expression of AtCPK1 leads to accumulation of salicylic acid (SA) and constitutive expression of SA‐regulated defence and disease resistance genes, which, in turn, results in broad‐spectrum protection against pathogen infection. Expression studies in mutants affected in SA‐mediated defence responses revealed an interlocked feedback loop governing AtCPK1 expression and components of the SA‐dependent signalling pathway. Moreover, we demonstrate the dual localization of AtCPK1 in lipid bodies and peroxisomes. Overall, our findings identify AtCPK1 as a component of the innate immune system of Arabidopsis plants.     

Role of ethylene signalling in growth and systemic resistance induction by the plant growth‐promoting fungus Penicillium viridicatum in Arabidopsis

The plant growth‐promoting fungi (PGPF) have long been known to improve plant growth and suppress plant diseases. The PGPF Penicillium viridicatum GP15‐1 elicited plant growth and induced systemic resistance (ISR) in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000 (Pst), leading to a restriction of pathogen growth and disease development. Examination of local and systemic genes indicated that GP15‐1 did not modulate the expression of any of the tested defence‐related marker genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene signalling pathways. Subsequent challenge of GP15‐1‐colonized plants with Pst bacterium primed Arabidopsis plants for enhanced activation of the JA‐inducible Atvsp (vegetative storage protein) gene at a later stage of infection. To assess the contribution of different signalling pathways in GP15‐1‐elicited plant growth and ISR, Arabidopsis genotypes implicated in SA signalling expressing the nahG transgene (NahG) or carrying disruption in NPR1 (npr1), JA signalling (jar1) and ethylene signalling (ein2) were tested. The GP15‐1‐induced plant growth and ISR were fully compromised in an ein2 mutation. Root colonization assay revealed that the inability of the ein2 mutant to express GP15‐1‐induced plant growth and ISR was not associated with reduced root colonization by GP15‐1. In conclusion, our results demonstrate the ethylene signalling pathway is involved in plant growth promotion and ISR elicitation by the PGPF P. viridicatum GP15‐1 in Arabidopsis. These results provide evidence that ethylene signalling has a substantial role in plant growth and disease resistance.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

The Elongator complex‐associated protein DRL1 plays a positive role in immune responses against necrotrophic fungal pathogens in Arabidopsis

DEFORMED ROOT AND LEAVES1 (DRL1) is an Arabidopsis homologue of the yeast TOXIN TARGET4 (TOT4)/KILLER TOXIN‐INSENSITIVE12 (KTI12) protein that is physically associated with the RNA polymerase II‐interacting protein complex named Elongator. Mutations in DRL1 and Elongator lead to similar morphological and molecular phenotypes, suggesting that DRL1 and Elongator may functionally overlap in Arabidopsis. We have shown previously that Elongator plays an important role in both salicylic acid (SA)‐ and jasmonic acid (JA)/ethylene (ET)‐mediated defence responses. Here, we tested whether DRL1 also plays a similar role as Elongator in plant immune responses. Our results show that, although DRL1 partially contributes to SA‐induced cytotoxicity, it does not play a significant role in SA‐mediated expression of PATHOGENESIS‐RELATED genes and resistance to the virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. In contrast, DRL1 is required for JA/ET‐ and necrotrophic fungal pathogen Botrytis cinerea‐induced defence gene expression and for resistance to B. cinerea and Alternaria brassicicola. Furthermore, unlike the TOT4/KTI12 gene which, when overexpressed in yeast, confers zymocin resistance, a phenotype of the tot4/kti12 mutant, overexpression of DRL1 does not change B. cinerea‐induced defence gene expression and resistance to this pathogen. Finally, DRL1 contains an N‐terminal P‐loop and a C‐terminal calmodulin (CaM)‐binding domain and is a CaM‐binding protein. We demonstrate that both the P‐loop and the CaM‐binding domain are essential for the function of DRL1 in B. cinerea‐induced expression of PDF1.2 and ORA59, and in resistance to B. cinerea, suggesting that the function of DRL1 in plant immunity may be regulated by ATP/GTP and CaM binding.     

Different mechanisms of Trichoderma virens‐mediated resistance in tomato against Fusarium wilt involve the jasmonic and salicylic acid pathways

In the present study, we investigated the role of Trichoderma virens (TriV_JSB100) spores or cell‐free culture filtrate in the regulation of growth and activation of the defence responses of tomato (Solanum lycopersicum) plants against Fusarium oxysporum f. sp. lycopersici by the development of a biocontrol–plant–pathogen interaction system. Two‐week‐old tomato seedlings primed with TriV_JSB100 spores cultured on barley grains (BGS) or with cell‐free culture filtrate (CF) were inoculated with Fusarium pathogen under glasshouse conditions; this resulted in significantly lower disease incidence in tomato Oogata‐Fukuju plants treated with BGS than in those treated with CF. To dissect the pathways associated with this response, jasmonic acid (JA) and salicylic acid (SA) signalling in BGS‐ and CF‐induced resistance was evaluated using JA‐ and SA‐impaired tomato lines. We observed that JA‐deficient mutant def1 plants were susceptible to Fusarium pathogen when they were treated with BGS. However, wild‐type (WT) BGS‐treated tomato plants showed a higher JA level and significantly lower disease incidence. SA‐deficient mutant NahG plants treated with CF were also found to be susceptible to Fusarium pathogen and displayed low SA levels, whereas WT CF‐treated tomato plants exhibited moderately lower disease levels and substantially higher SA levels. Expression of the JA‐responsive defensin gene PDF1 was induced in WT tomato plants treated with BGS, whereas the SA‐inducible pathogenesis‐related protein 1 acidic (PR1a) gene was up‐regulated in WT tomato plants treated with CF. These results suggest that TriV_JSB100 BGS and CF differentially induce JA and SA signalling cascades for the elicitation of Fusarium oxysporum resistance in tomato.     

A stress‐inducible sulphotransferase sulphonates salicylic acid and confers pathogen resistance in Arabidopsis

Sulphonation of small molecules by cytosolic sulphotransferases in mammals is an important process in which endogenous molecules are modified for inactivation/activation of their biological effects. Plants possess large numbers of sulphotransferase genes, but their biological functions are largely unknown. Here, we present a functional analysis of the Arabidopsis sulphotransferase AtSOT12 (At2g03760). AtSOT12 gene expression is strongly induced by salt, and osmotic stress and hormone treatments. The T‐DNA knock‐out mutant sot12 exhibited hypersensitivity to NaCl and ABA in seed germination, and to salicylic acid (SA) in seedling growth. In vitro enzyme activity assay revealed that AtSOT12 sulphonates SA, and endogenous SA levels suggested that sulphonation of SA positively regulates SA production. Upon challenging with the pathogen Pseudomonas syringae, sot12 mutant and AtSOT12 over‐expressing lines accumulate less and more SA, respectively, when compared with wild type. Consistent with the changes in SA levels, the sot12 mutant was more susceptible, while AtSOT12 over‐expressing plants are more resistant to pathogen infection. Moreover, pathogen‐induced PR gene expression in systemic leaves was significantly enhanced in AtSOT12 over‐expressing plants. The role of sulphonation of SA in SA production, mobile signalling and acquired systemic resistance is discussed.     

Genome‐wide association study reveals novel players in defense hormone crosstalk in Arabidopsis

Jasmonic acid (JA) regulates plant defenses against necrotrophic pathogens and insect herbivores. Salicylic acid (SA) and abscisic acid (ABA) can antagonize JA‐regulated defenses, thereby modulating pathogen or insect resistance. We performed a genome‐wide association (GWA) study on natural genetic variation in Arabidopsis thaliana for the effect of SA and ABA on the JA pathway. We treated 349 Arabidopsis accessions with methyl JA (MeJA), or a combination of MeJA and either SA or ABA, after which expression of the JA‐responsive marker gene PLANT DEFENSIN1.2 (PDF1.2) was quantified as a readout for GWA analysis. Both hormones antagonized MeJA‐induced PDF1.2 in the majority of the accessions but with a large variation in magnitude. GWA mapping of the SA‐ and ABA‐affected PDF1.2 expression data revealed loci associated with crosstalk. GLYI4 (encoding a glyoxalase) and ARR11 (encoding an Arabidopsis response regulator involved in cytokinin signalling) were confirmed by T‐DNA insertion mutant analysis to affect SA–JA crosstalk and resistance against the necrotroph Botrytis cinerea. In addition, At1g16310 (encoding a cation efflux family protein) was confirmed to affect ABA–JA crosstalk and susceptibility to Mamestra brassicae herbivory. Collectively, this GWA study identified novel players in JA hormone crosstalk with potential roles in the regulation of pathogen or insect resistance.     

Semi‐dominant mutation in the cysteine‐rich receptor‐like kinase gene,ALS1, conducts constitutive defence response in rice

• Plants have evolved a sophisticated two‐branch defence system to prevent the growth and spread of pathogen infection. The novel Cys‐rich repeat (CRR) containing receptor‐like kinases, known as CRKs, were reported to mediate defence resistance in plants. For rice, there are only two reports of CRKs. A semi‐dominant lesion mimic mutant als1 (apoptosis leaf and sheath 1) in rice was identified to demonstrate spontaneous lesions on the leaf blade and sheath.
• A map‐based cloning strategy was used for fine mapping and cloning of ALS1, which was confirmed to be a typical CRK in rice. Functional studies of ALS1 were conducted, including phylogenetic analysis, expression analysis, subcellular location and blast resistance identification.
• Most pathogenesis‐related (PR) genes and other defence‐related genes were activated and up‐regulated to a high degree. ALS1 was expressed mainly in the leaf blade and sheath, in which further study revealed that ALS1 was present in the vascular bundles. ALS1 was located in the cell membrane of rice protoplasts, and its mutation did not change its subcellular location. Jasmonic acid (JA) and salicylic acid (SA) accumulation were observed in als1, and enhanced blast resistance was also observed.
• The mutation of ALS1 caused a constitutively activated defence response in als1. The results of our study imply that ALS1 participates in a defence response resembling the common SA‐, JA‐ and NH1‐mediated defence responses in rice.

     

A 13‐lipoxygenase is Expressed Early in the Hypersensitive Reaction of Cotton Plants to Xanthomonas campestris pv. malvacearum

Lipoxygenases (LOXs) are enzymes responsible for lipid peroxidation processes during plant defence responses to pathogen infection. Jasmonates are lipid‐derived signals that mediate plant stress responses with chloroplastic LOXs implicated in the biosynthesis of oxylipins like jasmonic acid (JA). Hypersensitive reaction (HR) cell death of cotton to the incompatible race 18 of Xanthomonas campestris pathovar malvacearum (Xcm) is associated with 9S‐lipoxygenase activity and expression of a 9‐LOX GhLOX1. Here, we report the cloning of cotton (Gossypium hirsutum L.) LOX gene GhLOX2. Sequence analysis showed that GhLOX2 is a putative 13‐LOX with a chloroplast‐transit peptide in the amino acid terminus. GhLOX2 was found to be significantly expressed in the first hour of Xcm‐induced HR. Investigation into LOX signalization on cotyledons incubated with methyl‐jasmonate (MeJA) or infiltrated with salicylic acid (SA) or ethylene (ET) revealed that the first two treatments induced GhLOX2 gene expression. Our results show that GhLOX2 gene expression occurred at the stage of the HR prior biochemical events previously highlighted. The role that GhLOX2 may have in the defence strategy of cotton to Xcm is discussed regarding the HR.     

β-Aminobutyric Acid-induced Resistance in Brassica juncea against the Necrotrophic Pathogen Alternaria brassicae

β‐Aminobutyric acid (BABA) pretreatment of Brassica plants protected them against the necrotrophic pathogen Alternaria brassicae. The achieved resistance level was much higher than that seen after salicylic acid (SA) and jasmonic acid (JA) pretreatments. BABA pretreatment to the leaves, 1 day before inoculation, led to an inhibition of the oxidative burst and a decrease in SA levels, but did not influence lipoxygenase activity nor cause callose deposition at the site of inoculation. Expression of two marker genes of the SA and JA pathways, namely PR1 and PDF1.2, was enhanced in response to BABA pretreatment. Our results indicate that BABA‐induced resistance is mediated through an enhanced expression of pathogenesis‐related protein genes, independent of SA and JA accumulation.