Abscisic Acid–Responsive 18 (CaABR18) Protein from Chickpea Inhibits the Growth of the Wilt-Causing Fusarium oxysporum f. sp. ciceri Race1

Multigene family pathogenesis-related-10 (PR-10) proteins are indispensable for initiation of plant defense reactions upon pathogen attack. Here, we report the isolation and differential induction of Cicer arietinum L. ABR18 (CaABR18) gene in susceptible and resistant chickpea upon exposure to Fusarium oxysporum f. sp. ciceri Race1 (Foc1). Further, sequence analysis and structural studies confirmed that CaABR18 protein possesses conserved glycine-rich P-loop motif and Betv 1 domain, which are common to many PR-10 family proteins. CaABR18 gene was found to be expressed in all the developing organs, with higher abundance in the mature leaves. Foc1 inoculation resulted in higher expression of CaABR18 gene in the resistant chickpea compared with susceptible one. CaABR18 protein induction was also observed by salicylic acid (SA) or abscisic acid (ABA) treatment. Biochemical analysis was performed using in vitro purified histidine-tagged recombinant ABR18 protein. Purified recombinant protein exhibits in vitro RNase and DNase activities. Application of recombinant ABR18 protein increases PI/SYTOX green uptake and nuclear disintegration and suppresses the growth of Foc1 hyphae in vitro. Agrobacterium-mediated transient expression of ABR18-YFP triggers reactive oxygen species (ROS) formation and cell death in Nicotiana benthamiana leaves. The fusion protein is shown to be targeted to the host nucleus. Taken together, our results revealed that CaABR18 imparts Fusarium resistance in chickpea by RNA/DNA degradation within host cells leading to programmed cell death (PCD) and also shows antifungal activity through its proper internalization, increasing membrane permeability and nuclear disintegration of Foc1.

     

Early burst of reactive oxygen species positively regulates resistance of eggplant against bacterial wilt

Reactive oxygen species (ROS) play a crucial role in the early response to plant biotic and abiotic stresses. In this study, bacterial wilt‐resistant and wilt‐susceptible eggplants were inoculated with Ralstonia solanacearum and the ROS content was analysed. The result revealed an increased accumulation of hydrogen peroxide (H₂O₂) and superoxide (O₂^?) in resistant and susceptible eggplant roots after R. solanacearum inoculation. H₂O₂ and O₂^? accumulation increased earlier in the inoculated resistant eggplant root than in the inoculated susceptible eggplant root. Real‐time polymerase chain reaction results revealed that respiratory burst oxidase homologue (Rboh) A, RbohB, RbohF and PR1 expression levels increased in inoculated resistant eggplant roots at an early stage (0–60 h postinoculation) and were at higher expression levels than those in susceptible eggplant roots. Ascorbate peroxidase, peroxidase and catalase activities were higher in inoculated resistant eggplant roots than in susceptible eggplant roots at the early stage. Hence, an early ROS burst positively regulates bacterial wilt resistance in eggplant.     

A proteomic study of in-root interactions between chickpea pathogens: the root-knot nematode Meloidogyne artiellia and the soil-borne fungus Fusarium oxysporum f. sp. ciceris race 5

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. ciceris (Foc) is the main soil-borne disease limiting chickpea production. Management of this disease is achieved mainly by the use of resistant cultivars. However, co-infection of a Foc-resistant plant by the fungus and the root-knot nematode Meloidogyne artiellia (Ma) causes breakdown of the resistance and thus limits its efficacy in the control of Fusarium wilt. In this work we aimed to reveal key aspects of chickpea:Foc:Ma interactions, studying fungal- and nematode-induced changes in root proteins, using chickpea lines 'CA 336.14.3.0' and 'ICC 14216K' that show similar resistant (Foc race 5) and susceptible (Ma) responses to either pathogen alone but a differential response after co-infection with both pathogens. 'CA 336.14.3.0' and 'ICC 14216K' chickpea plants were challenged with Foc race 5 and Ma, either in single or in combined inoculations, and the root proteomes were analyzed by two-dimensional gel electrophoresis using three biological replicates. Pairwise comparisons of treatments indicated that 47 protein spots in 'CA 336.14.3.0' and 31 protein spots in 'ICC 14216K' underwent significant changes in intensity. The responsive protein spots tentatively identified by MALDI TOF-TOF MS (27 spots for 'CA 336.14.3.0' and 15 spots for 'ICC 14216K') indicated that same biological functions were involved in the responses of either chickpea line to Foc race 5 and Ma, although common as well as line-specific responsive proteins were found within the different biological functions. To the best of our knowledge, this is the first study at the root proteome level of chickpea response to a biotic stress imposed by single and joint infections by two major soil-borne pathogens.     

The sucrose transporter SlSUT2 from tomato interacts with brassinosteroid functioning and affects arbuscular mycorrhiza formation

Mycorrhizal plants benefit from the fungal partners by getting better access to soil nutrients. In exchange, the plant supplies carbohydrates to the fungus. The additional carbohydrate demand in mycorrhizal plants was shown to be balanced partially by higher CO₂ assimilation and increased C metabolism in shoots and roots. In order to test the role of sucrose transport for fungal development in arbuscular mycorrhizal (AM) tomato, transgenic plants with down‐regulated expression of three sucrose transporter genes were analysed. Plants that carried an antisense construct of SlSUT2 (SlSUT2as) repeatedly exhibited increased mycorrhizal colonization and the positive effect of plants to mycorrhiza was abolished. Grafting experiments between transgenic and wild‐type rootstocks and scions indicated that mainly the root‐specific function of SlSUT2 has an impact on colonization of tomato roots with the AM fungus. Localization of SISUT2 to the periarbuscular membrane indicates a role in back transport of sucrose from the periarbuscular matrix into the plant cell thereby affecting hyphal development. Screening of an expression library for SlSUT2‐interacting proteins revealed interactions with candidates involved in brassinosteroid (BR) signaling or biosynthesis. Interaction of these candidates with SlSUT2 was confirmed by bimolecular fluorescence complementation. Tomato mutants defective in BR biosynthesis were analysed with respect to mycorrhizal symbiosis and showed indeed decreased mycorrhization. This finding suggests that BRs affect mycorrhizal infection and colonization. If the inhibitory effect of SlSUT2 on mycorrhizal growth involves components of BR synthesis and of the BR signaling pathway is discussed.     

The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2

Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate pre‐symbiotic growth of the fungus, which releases lipochito‐oligosaccharides (Myc‐LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up‐regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc‐LCOs. Fungal colonization was much reduced by over‐expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc‐LCOs, that prevents over‐colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA‐mediated negative regulation of NSP2.     

Tracking changes in life‐history traits related to unnecessary virulence in a plant‐parasitic nematode

Evaluating trade‐offs in life‐history traits of plant pathogens is essential to understand the evolution and epidemiology of diseases. In particular, virulence costs when the corresponding host resistance gene is lacking play a major role in the adaptive biology of pathogens and contribute to the maintenance of their genetic diversity. Here, we investigated whether life‐history traits directly linked to the establishment of plant–nematode interactions, that is, ability to locate and move toward the roots of the host plant, and to invade roots and develop into mature females, are affected in Meloidogyne incognita lines virulent against the tomato Mi‐1.2 resistance gene. Virulent and avirulent near‐isogenic lines only differing in their capacity to reproduce or not on resistant tomatoes were compared in single inoculation or pairwise competition experiments. Data highlighted (1) a global lack of trade‐off in traits associated with unnecessary virulence with respect to the nematode ability to successfully infest plant roots and (2) variability in these traits when the genetic background of the nematode is considered irrespective of its (a)virulence status. These data suggest that the variation detected here is independent from the adaptation of M. incognita to host resistance, but rather reflects some genetic polymorphism in this asexual organism.     

Enhanced Defence Responses of Chickpea Plants Against Rhizoctonia solani by Pre‐Inoculation with Rhizobia

The aim of this work was to study the antagonist effect of two Rhizobium strains Pch Azm and Pch S.Nsir2 to Rhizoctonia solani and for an evaluation of the relative impact of rhizobia on the expression of the plant's defence response against Rhizoctonia. First, these strains reduced fungal growth observed in vitro using the same or separately Petri dishes. Moreover, these isolates led to reduced chickpea infection by R. solani, resulting from the direct effect of rhizobia on pathogens and possible induced resistance in chickpea. Concomitantly, reduction in infection was accompanied by enhanced level of defence‐related enzymes, phenylalanine ammonia lyase (PAL) and peroxidase (POX). An increased level of phenol content was recorded in the roots of bacterized plants grown in the presence of pathogen. The results promise the use of rhizobia for protection of chickpea against R. solani.     

‘Real time’ genetic manipulation: a new tool for ecological field studies

Field experiments with transgenic plants often reveal the functional significance of genetic traits that are important for the performance of the plants in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene‐related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression for the study of ecological interactions in real time, of genetic traits that play essential roles in development, or of dose‐dependent effects. We applied the sensitive dexamethasone (DEX)‐inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin‐based DEX application method to facilitate ectopic gene expression and RNA interference‐mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field‐grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CKs in both the glasshouse and the field to understand resistance to the native herbivore Tupiocoris notatus, which attacks plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, damage by T. notatus increased, demonstrating the role of CKs in this plant–herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature.     

Robust biological nitrogen fixation in a model grass–bacterial association

Nitrogen‐fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C₄ grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen‐13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen‐limiting conditions when inoculated with an ammonium‐excreting strain of Azospirillum brasilense. ¹¹C‐labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen‐starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen‐sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.     

An untargeted global metabolomic analysis reveals the biochemical changes underlying basal resistance and priming in Solanum lycopersicum,and identifies 1‐methyltryptophan as a metabolite involved in plant responses to Botrytis cinerea and Pseudomonas syringae

In this study, we have used untargeted global metabolomic analysis to determine and compare the chemical nature of the metabolites altered during the infection of tomato plants (cv. Ailsa Craig) with Botrytis cinerea (Bot) or Pseudomonas syringae pv. tomato DC3000 (Pst), pathogens that have different invasion mechanisms and lifestyles. We also obtained the metabolome of tomato plants primed using the natural resistance inducer hexanoic acid and then infected with these pathogens. By contrasting the metabolomic profiles of infected, primed, and primed + infected plants, we determined not only the processes or components related directly to plant defense responses, but also inferred the metabolic mechanisms by which pathogen resistance is primed. The data show that basal resistance and hexanoic acid‐induced resistance to Bot and Pst are associated with a marked metabolic reprogramming. This includes significant changes in amino acids, sugars and free fatty acids, and in primary and secondary metabolism. Comparison of the metabolic profiles of the infections indicated clear differences, reflecting the fact that the plant's chemical responses are highly adapted to specific attackers. The data also indicate involvement of signaling molecules, including pipecolic and azelaic acids, in response to Pst and, interestingly, to Bot. The compound 1‐methyltryptophan was shown to be associated with the tomato–Pst and tomato–Bot interactions as well as with hexanoic acid‐induced resistance. Root application of this Trp‐derived metabolite also demonstrated its ability to protect tomato plants against both pathogens.     

Changes in the redox status of chickpea roots in response to infection by Fusarium oxysporum f. sp. ciceris: apoplastic antioxidant enzyme activities and expression of oxidative stress-related genes

Activity levels of oxidative stress-related enzymes in the root apoplast during the interaction of WR315 (resistant) and JG62 (susceptible) chickpeas ( Cicer arietinum L.) with the highly virulent race 5 of Fusarium oxysporum f. sp. ciceris were compared. Because this fungus develops asymptomatic infections in the chickpea root cortex in both susceptible and resistant plants, but only intrudes into the root xylem in the susceptible variety, the interactions were compared at three specific stages during disease development in JG62: (i) before symptom development (10 days after inoculation); (ii) at the time of appearance of the first disease symptoms (15–17 days after inoculation) and (iii) when all plants had developed disease symptoms (20–22 days after inoculation). Diamine oxidase (DAO), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol-dependent peroxidase and superoxide dismutase (SOD), but not catalase (CAT), were found in the apoplast of chickpea roots. In terms of APX activity, infection by the pathogen caused a different response in the incompatible compared to the compatible plant. In the case of GR, SOD and DAO activities, the pathogen caused the same response, but it developed earlier ( i.e. GR and SOD) or to higher levels ( i.e. DAO) in the incompatible interaction. Expression of apx , cat , sod , lipoxygenase ( lox ) and actin genes was also analysed in infected roots. Infection by F. oxysporum f. sp. ciceris race 5 only caused a significant change in the root expression of lox and actin genes. This up-regulation was earlier ( lox ) or higher ( actin ) in the incompatible than in the compatible interaction. Thus, changes in oxidative metabolism differ in compatible and incompatible interactions in Fusarium wilt of chickpea.     

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.     

A role for β‐sitosterol to stigmasterol conversion in plant–pathogen interactions

Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana–Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from β‐sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen‐inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild‐type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild‐type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen‐associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/β‐sitosterol ratio as whole‐leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid‐mediated defence signalling but attenuate pathogen‐induced expression of the defence regulator flavin‐dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to β‐sitosterol ratio in plant membranes.     

Lotus SHAGGY‐like kinase 1 is required to suppress nodulation in Lotus japonicus

Glycogen synthase kinase/SHAGGY‐like kinases (SKs) are a highly conserved family of signaling proteins that participate in many developmental, cell‐differentiation, and metabolic signaling pathways in plants and animals. Here, we investigate the involvement of SKs in legume nodulation, a process requiring the integration of multiple signaling pathways. We describe a group of SKs in the model legume Lotus japonicus (LSKs), two of which respond to inoculation with the symbiotic nitrogen‐fixing bacterium Mesorhizobium loti. RNAi knock‐down plants and an insertion mutant for one of these genes, LSK1, display increased nodulation. Ηairy‐root lines overexpressing LSK1 form only marginally fewer mature nodules compared with controls. The expression levels of genes involved in the autoregulation of nodulation (AON) mechanism are affected in LSK1 knock‐down plants at low nitrate levels, both at early and late stages of nodulation. At higher levels of nitrate, these same plants show the opposite expression pattern of AON‐related genes and lose the hypernodulation phenotype. Our findings reveal an additional role for the versatile SK gene family in integrating the signaling pathways governing legume nodulation, and pave the way for further study of their functions in legumes.     

Small rubber particle proteins from Taraxacum brevicorniculatum promote stress tolerance and influence the size and distribution of lipid droplets and artificial poly(cis‐1,4‐isoprene) bodies

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex‐containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1–5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1–5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild‐type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3–5 on the dispersity of artificial poly(cis‐1,4‐isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet‐associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants.