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Drought plays a central role in increasing the incidence and severity of dry root rot (DRR) disease in chickpea. This is an economically devastating disease, compromising chickpea yields particularly severely in recent years due to erratic rainfall patterns. Macrophomina phaseolina (formerly Rhizoctonia bataticola) is the causal agent of DRR disease in the chickpea plant. The infection pattern in chickpea roots under well-watered conditions and drought stress are poorly understood at present. This study provides detailed disease symptomatology and the characteristics of DRR fungus at morphological and molecular levels. Using microscopy techniques, the infection pattern of DRR fungus in susceptible chickpea roots was investigated under well-watered and drought-stress conditions. Our observations suggested that drought stress intensifies the progression of already ongoing infection by weakening the endodermal barrier and overall defense. Transcriptomic analysis suggested that the plant’s innate immune defense program is downregulated in infected roots when subjected to drought stress. Furthermore, genes involved in hormonal regulation are differentially expressed under drought stress. These findings provide hints in terms of potential chickpea genes to target in crop improvement programs to develop climate-change-resilient cultivars. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Chickpea is an essential crop for protein nutrition and is grown around the world in rain-fed conditions. However, chickpea cultivation is under threat due to emerging diseases favored by drought stress. Dry root rot (DRR), an economically devastating disease, is an example. Chickpea-specific strains of a necrotic fungal phytopathogen, Macrophomina phaseolina, cause DRR. Microsclerotia of this fungus, which are capable of withstanding harsh environmental conditions, serve as primary inoculum. Initial symptoms are scattered necrotic spots in roots, progressing to rotting and withering lateral roots, accompanied by prematurely dried, straw-colored foliage. The recent rise in global temperature and worsening of drought spells have aggravated DRR outbreaks in chickpea. To date, DRR epidemiology has not been clarified in detail. Also, the literature lacks clarity on M. phaseolina taxonomy, morphology, disease progression, and diagnosis. In this article, research progress on patterns of DRR occurrence in the field and belowground and aboveground symptoms are clarified. In addition, the current understanding of taxonomy and management practices is elaborated. We also summarize knowledge of the impact of drought and high temperature on DRR severity. Furthermore, we provide future perspectives on the importance of host resistance, quantitative trait loci identification, and genotype screening for the identification of resistant genotypes. The article proposes new research priorities and a corresponding plan for the mitigation of DRR.

Many plant-encoded E3 ligases are known to be involved in plant defense. Here, we report a novel role of E3 ligase SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1) in plant immunity. Even though SDIR1 is reasonably well-characterized, its role in biotic stress response is not known. The silencing of SDIR1 in Nicotiana benthamiana reduced the multiplication of the virulent bacterial pathogen Pseudomonas syringae pv. tabaci. The Arabidopsis sdir1 mutant is resistant to virulent pathogens, whereas SDIR1 overexpression lines are susceptible to both host and nonhost hemibiotrophic bacterial pathogens. However, sdir1 mutant and SDIR1 overexpression lines showed hypersusceptibility and resistance, respectively, against the necrotrophic pathogen Erwinia carotovora. The mutant of SDIR1 target protein, i.e., SDIR-interacting protein 1 (SDIR1P1), also showed resistance to host and nonhost pathogens. In SDIR1 overexpression plants, transcripts of NAC transcription factors were less accumulated and the levels of jasmonic acid (JA) and abscisic acid were increased. In the sdir1 mutant, JA signaling genes JAZ7 and JAZ8 were downregulated. These data suggest that SDIR1 is a susceptibility factor and its activation or overexpression enhances disease caused by P. syringae pv. tomato DC3000 in Arabidopsis. Our results show a novel role of SDIR1 in modulating plant defense gene expression and plant immunity. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

We report an advanced web server, the plant-specific small noncoding RNA interference tool pssRNAit, which can be used to design a pool of small interfering RNAs (siRNAs) for highly effective, specific, and nontoxic gene silencing in plants. In developing this tool, we integrated the transcript dataset of plants, several rules governing gene silencing, and a series of computational models of the biological mechanism of the RNA interference (RNAi) pathway. The designed pool of siRNAs can be used to construct a long double-strand RNA and expressed through virus-induced gene silencing (VIGS) or synthetic transacting siRNA vectors for gene silencing. We demonstrated the performance of pssRNAit by designing and expressing the VIGS constructs to silence Phytoene desaturase (PDS) or a ribosomal protein-encoding gene, RPL10 (QM), in Nicotiana benthamiana. We analyzed the expression levels of predicted intended-target and off-target genes using reverse transcription quantitative PCR. We further conducted an RNA-sequencing-based transcriptome analysis to assess genome-wide off-target gene silencing triggered by the fragments that were designed by pssRNAit, targeting different homologous regions of the PDS gene. Our analyses confirmed the high accuracy of siRNA constructs designed using pssRNAit. The pssRNAit server, freely available at https://plantgrn.noble.org/pssRNAit/, supports the design of highly effective and specific RNAi, VIGS, or synthetic transacting siRNA constructs for high-throughput functional genomics and trait improvement in >160 plant species.

Alternaria blight, caused by Alternaria brassicae, causes considerable yield loss in Brassica crops. While several blight-resistant varieties have been developed using resistance sources from host germplasm, none of them are entirely successful in imparting durable resistance. This has prompted the exploration of novel gene pools of nonhost plant species. Nonhost resistance (NHR) is a durable form of resistance, comprising pre- and postinvasion layers of defense. We aimed to identify the molecular basis of NHR to A. brassicae and identify the layers of NHR operating in a nonhost, chickpea (Cicer arietinum). To elucidate the layers of NHR operating against A. brassicae, we compared the histopathology and infection patterns of A. brassicae in C. arietinum and Brassica juncea. Delayed conidial germination, impeded hyphal growth, suppressed appressorium formation, and limited hyphal penetration occurred in the nonhost plant compared with the host plant, implying the involvement of the preinvasion layer of NHR in C. arietinum. Next, we investigated the molecular basis of robust NHR, in C. arietinum challenged with A. brassicae, by microarray-based global transcriptome profiling. Genes involved in stomatal closure, cuticular wax biosynthesis, cell-wall modification, and secondary metabolite production (contributing to preinvasion NHR) as well as reactive oxygen species (ROS) and cell death (contributing to postinvasion NHR) were found to be upregulated. Consistent with transcriptomic analysis, the morpho-pathological analysis revealed stomatal closure, ROS accumulation, and localized cell death in C. arietinum as the defense strategies against A. brassicae. Thus, we identified NHR-contributing genes with potential applications in blight resistance gene transfer to B. juncea.

Climate change has led to an increased number of abiotic and biotic stresses, and the plants experience these stresses simultaneously. Abiotic stresses like drought, heat, and salinity greatly influence plant-pathogen interaction when co-occurring with the biotic stressors. Recent studies have shown that combined stresses induce unique physiological and molecular responses, which involve rewiring of the hormonal pathways, accumulation of various metabolites and induction or suppression of immunity genes in plants. The net impact of the interaction, which depends on a multitude of factors, thus, modulates the effect of biotic stressors on plants by either increasing or decreasing plants susceptibility towards them. The present review aims to provide an overview of the current knowledge on the biotic and abiotic stress interactions in plants. We have discussed the role of drought, salt, and heat stress in influencing pathogen infection in plants in brief. Plants responses to the three types of combined stresses are compared to decipher the common and unique plant responses to these stresses.

A host-pathogen surpasses the plant defense machinery and successfully infects the plant to serve its needs. In contrast, a nonhost pathogen is restricted by plant immune responses. In this study, we deciphered the differential responses of Arabidopsis against the host-pathogen (Pseudomonas syringae pv. maculicola, Psm) and the nonhost pathogen (P. syringae pv. tabaci, Pst) infection. The Pst multiplication was restricted in Arabidopsis plant coinciding with the absence of any disease symptoms which was also associated with the increase in defense associated, pathogenesis related gene1 (PR1) expression and callose deposition. Host-pathogen infection, on the other hand, caused chlorotic symptoms with much less activation of the defense marker genes compared to the nonhost infection. Proline content was decreased in plants infected with Psm but not in case of Pst infection. Proline is a crucial plant metabolite, and osmolyte suggested to be involved in plant defense responses. However, the differential regulation of proline pathway under a susceptible or resistance response has not yet been unravelled. The expression profile of proline metabolism genes in a time course post-infection revealed drastic differences after Psm and Pst pathogen infection. The elevated expression of proline catabolic genes, AtProDH and AtP5CDH were noted under Psm infection. In contrast, plants infected with Pst showed upregulation of AtProDH but a decline in AtP5CDH transcripts along with an upregulation of the biosynthesis genes i.e. AtP5CS and AtP5CR. Our study shows that proline metabolism is tightly regulated under host and nonhost pathogen infection and impacts susceptibility and resistance of the plants, respectively.

Sugar will eventually be exported transporters (SWEETs), a newly discovered class of sugar transporters, play a significant role in sugar efflux processes across various kingdoms of life. In fact, SWEETs have a long evolutionary path from prokaryotes to higher plants. In plants, they are involved in developmental processes, including nectar secretion, pollen nutrition, and seed filling. While the role of SWEETs has been well studied in biotic stresses, particularly their manipulation by pathogens for sugar acquisition, they have also been linked to many abiotic stresses. Although the phylogenetic relationships and solved structures of SWEETs in different plants have been revealed, their regulation remains unexplored. The current review deals with all the exciting discoveries around SWEETs, including their classification and diversity, and bridges the gaps in their evolutionary story, from bacterial semiSWEETs to eukaryotic SWEETs. We also critically examine SWEETs at genomic, transcriptomic, and proteomic levels, as evinced by recently published examples from grain, millet, and horticultural crops. In addition, we highlight the possibilities of utilizing SWEETs in applications such as bioethanol production and disease diagnostic markers. We attempt to elucidate and unify findings related to the yet unsolved puzzle of SWEET regulation in plants to improve crop production and protection for sustainable agriculture.

Depriving bacterial pathogens of sugars is a potential plant defense strategy. The relevance of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS (SWEETs) in plant susceptibility to pathogens has been established, but their role in plant defense remains unknown. We identified Arabidopsis thaliana SWEETs (AtSWEETs) involved in defense against nonhost and host Pseudomonas syringae pathogens through reverse genetic screening of atsweet1–17 mutants. Double/triple mutant, complementation, and overexpression line analysis, and apoplastic sucrose estimation studies revealed that AtSWEET12 suppresses pathogen multiplication by limiting sucrose availability in the apoplast. Localization studies suggested that plant defense occurred via increased plasma membrane targeting of AtSWEET12 with concomitant AtSWEET11 protein reduction. Moreover, the heterooligomerization of AtSWEET11 and AtSWEET12 was involved in regulating sucrose transport. Our results highlight a PAMP-mediated defense strategy against foliar bacterial pathogens whereby plants control AtSWEET11-mediated sucrose efflux in the apoplast through AtSWEET12. We uncover a fascinating new mechanism of pathogen starvation as a broad-spectrum disease resistance mechanism in parallel with existing immune pathways.One sentence summaryThe transporter AtSWEET12 restricts bacterial pathogen multiplication by regulating sucrose availability to pathogens in the apoplast.

Drought and leaf blight caused by the fungus Ascochyta rabiei often co-occur in chickpea (Cicer arietinum)-producing areas. While the responses of chickpea to either drought or A. rabiei infection have been extensively studied, their combined effect on plant defense mechanisms is unknown. Fine modulation of stress-induced signaling pathways under combined stress is an important stress adaptation mechanism that warrants a better understanding. Here we show that drought facilitates resistance against A. rabiei infection in chickpea. The analysis of proline levels and gene expression profiling of its biosynthetic pathway under combined drought and A. rabiei infection revealed the gene encoding proline dehydrogenase (CaProDH2) as a strong candidate conferring resistance to A. rabiei infection. Transcript levels of CaProDH2, pyrroline-5-carboxylate (P5C) quantification, and measurement of mitochondrial reactive oxygen species (ROS) production showed that fine modulation of the proline–P5C cycle determines the observed resistance. In addition, CaProDH2-silenced plants lost basal resistance to A. rabiei infection induced by drought, while overexpression of the gene conferred higher resistance to the fungus. We suggest that the drought-induced accumulation of proline in the cytosol helps maintain cell turgor and raises mitochondrial P5C contents by a CaProDH2-mediated step, which results in ROS production that boosts plant defense responses and confers resistance to A. rabiei infection. Our findings indicate that manipulating the proline–P5C pathway may be a possible strategy for improving stress tolerance in plants suffering from combined drought and A. rabiei infection.

Plant viruses encounter a range of host defenses including non-host resistance (NHR), leading to the arrest of virus replication and movement in plants. Viruses have limited host ranges, and adaptation to a new host is an atypical phenomenon. The entire genotypes of plant species which are imperceptive to every single isolate of a genetically variable virus species are described as non-hosts. NHR is the non-specific resistance manifested by an innately immune non-host due to pre-existing and inducible defense responses, which cannot be evaded by yet-to-be adapted plant viruses. NHR-to-plant viruses are widespread, but the phenotypic variation is often not detectable within plant species. Therefore, molecular and genetic mechanisms of NHR need to be systematically studied to enable exploitation in crop protection. This article comprehensively describes the possible mechanisms of NHR against plant viruses. Also, the previous definition of NHR to plant viruses is insufficient, and the main aim of this article is to sensitize plant pathologists to the existence of NHR to plant viruses and to highlight the need for immediate and elaborate research in this area.

Drought stress and pathogen infection simultaneously occur in the field. In this study, the interaction of these two stresses with chickpea, their individual and combined effect and the net impact on plant growth and yield traits were systematically assessed under field and confined pot experiments. The field experiments were conducted for four consecutive years from 2014–15 to 2017–18 at different locations of India. Different irrigation regimes were maintained to impose mild to severe drought stress, and natural incidence of the pathogen was considered as pathogen stress. We observed an increased incidence of fungal diseases namely, dry root rot (DRR) caused by Rhizoctonia bataticola, black root rot (BRR) caused by Fusarium solani under severe drought stress compared to well-irrigated field condition. Similar to field experiments, pot experiments also showed severe disease symptoms of DRR and BRR in the presence of drought compared to pathogen only stress. Overall, the results from this study not only showed the impact of combined drought and DRR stress but also provided systematic data, first of its kind, for the use of researchers.

Plants are naturally resistant to most pathogens through a broad and durable defense response called nonhost disease resistance. Nonhost disease resistance is a complex process that includes preformed physical and chemical barriers and induced responses. In spite of its importance, many components of nonhost disease resistance remain to be identified and characterized. Using virus-induced gene silencing in Nicotiana benthamiana, we discovered a novel gene that we named NbNHR2 (N. benthamiana nonhost resistance 2). NbNHR2-silenced plants were susceptible to the nonadapted pathogen Pseudomonas syringae pv. tomato T1, which does not cause disease in wild-type or nonsilenced N. benthamiana plants. We found two orthologous genes in Arabidopsis thaliana: AtNHR2A and AtNHR2B. Similar to the results obtained in N. benthamiana, Atnhr2a and Atnhr2b mutants were susceptible to the nonadapted bacterial pathogen of A. thaliana, P. syringae pv. tabaci. We further found that these mutants were also defective in callose deposition. AtNHR2A and AtNHR2B fluorescent protein fusions transiently expressed in N. benthamiana localized predominantly to chloroplasts and a few unidentified dynamic puncta. RFP-AtNHR2A and AtNHR2B-GFP displayed overlapping signals in chloroplasts, indicating that the two proteins could interact, an idea supported by coimmunoprecipitation studies. We propose that AtNHR2A and AtNHR2B are new components of a chloroplast-signaling pathway that activates callose deposition to the cell wall in response to bacterial pathogens.

The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components-plant, pathogen, and environment-involved in the disease triangle. Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

Premise We developed a systematic protocol for the easy, high-throughput, qualitative, and quantitative assessment of the patho-morphological, physiological, and molecular responses of Arabidopsis thaliana plants simultaneously subjected to drought and bacterial infection. This approach will assist studies elucidating plant adaptation strategies to combat combined stresses. Methods and results Plants were grown in small screw-capped containers, individual pots, or pot strips. Watering was withheld from 30-day-old plants, which were subsequently infected with Pseudomonas syringae pv. tomato DC3000 using a dip inoculation. The natural development of both drought and bacterial infection was successfully and rapidly replicated in large numbers of plants, which is difficult to achieve with existing protocols. Conclusions Our protocol offers a simple, low-cost, high-throughput strategy for the rapid and easy bacterial infection of large numbers of plants. It can be used in large-scale mutant and ecotype screenings under combined stresses and for other foliar pathogens in different plant species.

Sugar will eventually be exported transporters (SWEETs), a novel family of sugar transporters found in both eukaryotes and prokaryotes, facilitate sugar flux across the cell membrane. Although these transporters were first discovered in plants, their homologs have been reported in different organisms. SWEETs have critical roles in various developmental processes, including phloem loading, nectar secretion, and pathogen nutrition. The structure of bacterial homologs, called SemiSWEETs, has been well studied thus far. Here, we provide an overview of SWEET protein structure and dynamic function by analyzing the solved crystal structures and predicted models that are available for a few SWEETs in a monocot plant (rice) and dicot plant (Arabidopsis thaliana). Despite the advancement in structure-related studies, the regulation of SWEETs remains unknown. In light of reported regulatory mechanisms of a few other sugar transporters, we propose the regulation of SWEETs at the post-translational level. We then enumerate the potential post-translational modification sites in SWEETs using computational tools. Overall, in this review, we critically analyze SWEET protein structure in plants to predict the post-translational regulation of SWEETs. Such findings have a direct bearing on plant nutrition and defense and targeting the regulation at these levels will be important in crop improvement.

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In this study, the metabolic enzyme formate dehydrogenase (FDH1) was identified to be involved in nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. Arabidopsis Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that SA is most likely involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to only mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 localized to mitochondria and the amount of FDH1 localized to mitochondria increased upon infection with host or nonhost pathogens. Interestingly, the subcellular localization of FDH1 was observed in both mitochondria and chloroplasts after infection with a nonhost pathogen in Arabidopsis. We speculate that FDH1 plays a role in cellular signaling networks between mitochondria and chloroplasts to produce coordinated defense responses such as SA-induced reactive oxygen species (ROS) generation and hypersensitive response (HR)-induced cell death against nonhost bacterial pathogens.

Ribosomes play an integral part in plant growth, development, and defence responses. We report here the role of ribosomal protein large (RPL) subunit QM/RPL10 in nonhost disease resistance. The RPL10‐silenced Nicotiana benthamiana plants showed compromised disease resistance against nonhost pathogen Pseudomonas syringae pv. tomato T1. The RNA‐sequencing analysis revealed that many genes involved in defence and protein translation mechanisms were differentially affected due to silencing of NbRPL10. Arabidopsis AtRPL10 RNAi and rpl10 mutant lines showed compromised nonhost disease resistance to P. syringae pv. tomato T1 and P. syringae pv. tabaci. Overexpression of AtRPL10A in Arabidopsis resulted in reduced susceptibility against host pathogen P. syringae pv. tomato DC3000. RPL10 interacts with the RNA recognition motif protein and ribosomal proteins RPL30, RPL23, and RPS30 in the yeast two‐hybrid assay. Silencing or mutants of genes encoding these RPL10‐interacting proteins in N. benthamiana or Arabidopsis, respectively, also showed compromised disease resistance to nonhost pathogens. These results suggest that QM/RPL10 positively regulates the defence and translation‐associated genes during nonhost pathogen infection., Ribosomal protein QM/RPL10 and its interacting proteins RRM, RPL30, RPL23, and RPS30 have extraribosomal functions and play a role in nonhost disease resistance.

Plants have complex and adaptive innate immune responses against pathogen infections. Stomata are key entry points for many plant pathogens. Both pathogens and plants regulate stomatal aperture for pathogen entry and defense, respectively. Not all plant proteins involved in stomatal aperture regulation have been identified. Here, we report GENERAL CONTROL NONREPRESSIBLE4 (GCN4), an AAA+-ATPase family protein, as one of the key proteins regulating stomatal aperture during biotic and abiotic stress. Silencing of GCN4 in Nicotiana benthamiana and Arabidopsis thaliana compromises host and nonhost disease resistance due to open stomata during pathogen infection. AtGCN4 overexpression plants have reduced H+-ATPase activity, stomata that are less responsive to pathogen virulence factors such as coronatine (phytotoxin produced by the bacterium Pseudomonas syringae) or fusicoccin (a fungal toxin produced by the fungus Fusicoccum amygdali), reduced pathogen entry, and enhanced drought tolerance. This study also demonstrates that AtGCN4 interacts with RIN4 and 14-3-3 proteins and suggests that GCN4 degrades RIN4 and 14-3-3 proteins via a proteasome-mediated pathway and thereby reduces the activity of the plasma membrane H+-ATPase complex, thus reducing proton pump activity to close stomata.

Drought transcriptome analysis of finger millet (Eleusine coracana) by cDNA subtraction identified drought responsive genes that have a potential role in drought tolerance. Through virus-induced gene silencing (VIGS) in a related crop species, maize (Zea mays), several genes, including a G-BOX BINDING FACTOR 3 (GBF3) were identified as candidate drought stress response genes and the role of GBF3 in drought tolerance was studied in Arabidopsis thaliana. Overexpression of both EcGBF3 and AtGBF3 in A. thaliana resulted in improved tolerance to osmotic stress, salinity and drought stress in addition to conferring insensitivity to ABA. Conversely, loss of function of this gene increased the sensitivity of A. thaliana plants to drought stress. EcGBF3 transgenic A. thaliana results also suggest that drought tolerance of sensitive plants can be improved by transferring genes from far related crops like finger millet. Our results demonstrate the role of GBF3 in imparting drought tolerance in A. thaliana and indicate the conserved role of this gene in drought and other abiotic stress tolerance in several plant species.

Field-grown plants experience cycles of drought stress and recovery due to variation in soil moisture status. Physiological, biochemical and transcriptome responses instigated by recovery are expected to be different from drought stress and non-stressed state. Such responses can further aid or antagonize the plant’s interaction with the pathogen. However, at molecular level, not much is known about plant-pathogen interaction during drought recovery. In the present study, we performed a microarray-based global transcriptome profiling and demonstrated the existence of unique transcriptional changes in Arabidopsis thaliana inoculated with Pseudomonas syringae pv. tomato DC3000 at the time of drought recovery (drought recovery pathogen, DRP) when compared to the individual drought (D) or pathogen (P) or drought recovery (DR). Furthermore, the comparison of DRP with D or DR and P transcriptome revealed the presence of a few common genes among three treatments. Notably, a gene encoding proline dehydrogenase (AtProDH1) was found to be commonly up-regulated under drought recovery (DR), DRP and P stresses. We also report an up-regulation of pyrroline-5-carboxylate biosynthesis pathway during recovery. We propose that AtProDH1 influences the defense pathways during DRP. Altogether, this study provides insight into the understanding of defense responses that operate in pathogen-infected plants during drought recovery.

Plants in the natural conditions are often challenged by a combination of two or more stressors. A combination of drought and pathogen is one of the most pressing threats to the plant’s growth and survival in the field, and thus warrants a mechanistic understanding. Susceptible plant-pathogen interaction, owing to effector-mediated suppression of plant defense responses, limits its scope for combined stress studies. In the present study, we have investigated the morpho-physiological responses of Arabidopsis thaliana to simultaneous drought and nonhost bacterial pathogen Pseudomonas syringae pv. tabaci. Combined stress treatment provoked an early and more pronounced hypersensitive response in the plant as compared to the non-host pathogen treatment. We have further deciphered the molecular basis for the robust defense response observed under combined stress by transcriptomic profiling carried out using whole-genome microarray. We found that the enhanced resistance to the combined stress is accompanied by a massive transcriptional reprogramming involving several transcripts specifically responding to the stress combination. A prominent over-representation of genes involved in basal defense-related machinery was observed under the combined stress. Genes involved in various defense signaling cascades, accumulation of secondary metabolites and those encoding for receptor-like kinases were highly up-regulated under the combined stress. Up-regulated genes related to redox homeostasis and hypersensitive response (HR)-mediated cell death were also found to be markedly enriched under combined stress. We also compared the global gene expression profile of A. thaliana subjected to combined drought-nonhost bacteria to those treated with a combination of drought-host bacteria Pseudomonas syringae pv. tomato DC3000. A significant induction of genes responding to drought as well as bacteria was observed during both the interactions. However, the amplitude of induction was more pronounced under the combination of drought and nonhost bacteria. Our results also indicate that plant activates multiple defense pathways upon exposure to combined stress which strengthens the overall basal immunity of the plant, characterized by a stronger HR response.

In nature, plants and microorganisms interact constantly to modulate their growth and development. The interaction could be beneficial or hostile. Plants are constantly exposed to a range of pathogens, and have evolved mechanisms to fight the pathogen infections with the help of complex networking pathways. In addition to the pathogens, many beneficial organisms such as endophytes, of both, bacterial and fungal origin, are ubiquitously present in all plants. Unlike from their pathogenic counterparts, the endophytes do not cause any disease symptoms in their host plants. Some of the recent scientific discoveries and views concerning plant–microbe interactions are presented here.

Premise A portable, simple, yet efficient method was developed for the rapid extraction of xylem sap from the stems and petioles of tomato plants for diagnostic and quantification assays of the xylem-colonizing wilt bacterium Ralstonia solanacearum. Methods and results Xylem saps were extracted from tomato stem sections using negative pressure generated from handheld needleless syringes. The samples were collected from plants grown under different soil moisture levels at four days after inoculation with the pathogen. Pipette tips were modified to serve as adapters for the stem sections. The quantification of the bacterial load in the extracted sap was performed by plating sap dilutions in Kelman's triphenyltetrazolium chloride (TTC) medium. Pathogen identity was further confirmed by performing a PCR using R. solanacearum-specific primers. Conclusions Due to its simplicity, portability, and thoroughness of extraction from predetermined tissue sizes, the method can potentially facilitate high-throughput onsite sampling from a large number of samples in a short time, which cannot be achieved with other available techniques.

BackgroundAlternaria brassicae, the causal organism of Alternaria blight, is a necrotroph infecting crops of theBrassicaceaefamily at all growth stages. To circumvent this problem, several disease management strategies are being used in the field, and disease-resistant varieties have also been developed. However, no strategy has proven completely successful, owing to the high variability in virulence amongA. brassicaeisolates, which causes a diverse spectrum of symptoms. Nonhost resistance (NHR) is a robust and broad-spectrum defense mechanism available in plants, and the exploitation of gene pools from plant species that are nonhost toA. brassicaecould serve as novel sources of resistance.MethodologyWe searched the literature using key words relevant to this study in various search engines, such as PubMed, Web of Science, and Google Scholar, as well as certain journal websites. The literature was retrieved, sorted, and mined to extract data pertinent to the present review.ResultsIn this review, we have comprehensively covered the recent progress made in developing Alternaria blight resistance inBrassicacrops by exploiting host germplasm. We also enumerate the potential NHR sources available forA. brassicaeand the NHR layers possibly operating against this pathogen. In addition, we propose different strategies for identifying NHR-related genes from nonhost plants and testing their relevance in imparting broad-spectrum resistance when transferred to host plants.ConclusionThis review will help broaden the current knowledge base pertaining to the resistance sources available in host germplasm, the exploitation of NHR mechanisms, and their applications in protectingBrassicacrops from Alternaria blight. The insights might also be applicable to a wider repertoire of plant pathogens.

Due to climatic changes, rice crop is affected by moisture deficit stress and pathogens. Tissue water limitation besides reducing growth rates, also renders the crop susceptible to the infection by Xanthomonas oryzae pv. oryzae (Xoo) that causes bacterial leaf blight. Independently, both drought adaptation and Xoo resistance have been extensively studied. Though the cross-talk between drought and Xoo stress responses have been explored from individual stress studies, examining the combinatorial stress response is limited in rice. Recently published combined stress studies showed that under the combined stress, maintenance of carbon assimilation is hindered and such response is regulated by overlapping cellular mechanisms that are different from either of the individual stresses. Several receptors, MAP kinases, transcription factors, and ribosomal proteins, are predicted for playing a role in cellular homeostasis and protects cells from combined stress effects. Here we provide a critical analysis of these aspects using information from the recently published combined stress literature. This review is useful for researchers to comprehend combinatorial stress response of rice plants to drought and Xoo.

Plants under combined stresses exhibit a prominent shift in molecular responses compared with plants exposed to the same stresses independently. Profiling responses to individual and combined stressors at the gene expression level have identified several genes with intersecting responses to these stressors. However, the upstream regulators at the intersection of plant responses to individual and combined stresses are not known. Here, using the transcriptome of Arabidopsis thaliana under individual and combined drought and Pseudomonas syringae infection, we identified several genes whose expression overlaps between individual and combined stresses. To study the key regulator of such an overlapping gene, we predicted that the expression of 1-Pyrroline-5-carboxylate synthase 1 (AtP5CS1) is regulated by ath-miR164c at post-transcriptional level. Our results from the stem-loop RT-PCR based expression analysis revealed significant downregulation of ath-miR164c in response to P. syringae infection under both well-irrigated (pathogen only) and drought stress (combined stress) conditions. Furthermore, an Arabidopsis loss-of-function mutant of the miRNA ath-miR164c exhibited resistance to pathogen infection under combined stress, unlike the wild-type plants, implicating the role of ath-miR164c in regulating plant immunity. AtP5CS1 gene expression and proline accumulation were enhanced in the ath-miR164c mutant plants relative to the wild-type plants, demonstrating that ath-miR164c regulates AtP5CS1 of the proline biosynthesis pathway, which was also validated by 5’RLM-RACE results. This miRNA-mediated modulation of AtP5CS1 gene expression under combined stress fills crucial gaps in identifying the key convergent players in the current understanding of plant stress responses.

In field conditions, plants are often exposed to a combination of abiotic and biotic stresses, for instance, drought and pathogen infection. Transcriptome studies on Arabidopsis thaliana and other plants under individual and combined drought and pathogen stresses have unveiled the activation of shared molecular defense mechanisms. These shared plant responses are characterized by commonly regulated genes under both individual as well as combined stresses. Therefore, the identification of commonly regulated genes during individual and combined stress conditions can reveal plant responses towards combined stress. Available transcriptome studies on combined-stressed plants have hinted at G-Box Binding Factor 3 (GBF3) as one of the regulatory components of the shared response. However, the mechanistic understanding of the role of AtGBF3 under combined drought and pathogen stress is not yet decoded. In the current study, we used genetic approaches to identify the role of AtGBF3 in conferring tolerance to individual and combined drought and pathogen stress. Atgbf3 mutant plants showed increased susceptibility, while AtGBF3-overexpressing plants were tolerant under individual and combined drought and Pseudomonas syringae pv. tomato infection stresses as compared to wild-type plants. We further analyzed the global transcriptome of Atgbf3 mutant plants under combined stress to identify its downstream targets. We also established a high-throughput method to apply combined polyethylene glycol and pathogen stress on Murashige and Skoog medium-grown plants to further validate the role of AtGBF3 in combined stress.

Virus-induced gene silencing (VIGS) is an important forward and reverse genetics method for the study of gene function in many plant species, especially Nicotiana benthamiana. However, despite the widespread use of VIGS, a searchable database compiling the phenotypes observed with this method is lacking. Such a database would allow researchers to know the phenotype associated with the silencing of a large number of individual genes without experimentation. We have developed a VIGS phenomics and functional genomics database (VPGD) that has DNA sequence information derived from over 4,000 N. benthamiana VIGS clones along with the associated silencing phenotype for approximately 1,300 genes. The VPGD has a built-in BLAST search feature that provides silencing phenotype information of specific genes. In addition, a keyword-based search function could be used to find a specific phenotype of interest with the corresponding gene, including its Gene Ontology descriptions. Query gene sequences from other plant species that have not been used for VIGS can also be searched for their homologs and silencing phenotype in N. benthamiana. VPGD is useful for identifying gene function not only in N. benthamiana but also in related Solanaceae plants such as tomato and potato. The database is accessible at http://vigs.noble.org. Noble Research Institute and NSF IOS-1025642

Virus‐induced gene silencing (VIGS) is an important forward and reverse genetics method for the study of gene function in many plant species, especially Nicotiana benthamiana. However, despite the widespread use of VIGS, a searchable database compiling the phenotypes observed with this method is lacking. Such a database would allow researchers to know the phenotype associated with the silencing of a large number of individual genes without experimentation. We have developed a VIGS phenomics and functional genomics database (VPGD) that has DNA sequence information derived from over 4,000 N. benthamiana VIGS clones along with the associated silencing phenotype for approximately 1,300 genes. The VPGD has a built‐in BLAST search feature that provides silencing phenotype information of specific genes. In addition, a keyword‐based search function could be used to find a specific phenotype of interest with the corresponding gene, including its Gene Ontology descriptions. Query gene sequences from other plant species that have not been used for VIGS can also be searched for their homologs and silencing phenotype in N. benthamiana. VPGD is useful for identifying gene function not only in N. benthamiana but also in related Solanaceae plants such as tomato and potato. The database is accessible at http://vigs.noble.org.

Crops in field conditions are challenged by the simultaneous occurrence of drought and pathogen stress. In the past, research was primarily focused on studying the impact of individual stresses on plants and selection of crop varieties potentially tolerant to particular stress by yield-associated morpho-physiological traits. However, several molecular responses of crop plants underlying morpho-physiological features to concurrent stresses are not similar to that of individual stresses. Certain morpho-physiological traits such as cell membrane stability, leaf water potential, stomatal movement, and root length were shown to be altered distinctly under combined stress to combat the stress condition. However, the relevance of such traits under combined stress tolerance is not precisely known. In this chapter, from the extensive literature survey, we identified several morpho-physiological changes that could be cognate with better plant performance under combined stress and represented them as traits that have potential to impart combined stress tolerance. We have comprehensively explained physiological and molecular basis for each trait and, where possible, suggested the ways to exploit the information for identification of varieties with prospective traits. Also, we proposed the need for systematically studying the underlying regulatory traits under combined stress conditions in the future.

Dissecting plant responses to individual stress factors has been one of the most intriguing areas of plant stress biology. However, plants growing in natural conditions deal with several stresses simultaneously, and respond to single and combined stressors in remarkably different manners. The tomato (Solanum lycopersicum) is one of the most important vegetable crops that is largely impacted by soil moisture deficit and wilt disease, and it provides an ideal system for understanding combined stress responses. However, to date, the combined effect of these two stressors on tomato plants has not been studied. Wilt pathogens rely on the presence of sufficient moisture for their pathogenicity. Tomato plants’ response to low soil moisture is shaped by several vital morpho-physiological changes. At the same time, it is hypothesized that wilt-resistant tomato genotypes often develop vascular systems that are anatomically akin to those in low soil moisture-resilient plants. In this chapter, we examine unique responses of tomato plants to drought and wilt disease to comprehend an inherent common defense program operational under combined stressors in tomato plants. We review the influence of different soil moisture levels on the prevalence and pathogenicity of wilt pathogens, with a particular focus on the xylem colonizing bacterium, Ralstonia solanacearum.

In nature, plants are simultaneously exposed to a combination of biotic and abiotic stresses that limit crop yields. Only recently, researchers have started understanding the molecular basis of combined biotic and abiotic stress interactions. Evidences suggest that under combined stress plants exhibit tailored physiological and molecular responses, in addition to several shared responses as part of their stress tolerance strategy. These tailored responses are suggested to occur only in plants exposed to simultaneous stresses and this information cannot be inferred from individual stress studies. In this review article, we provide update on the responses of plants to simultaneous biotic and abiotic stresses, in particular drought and pathogen. Simultaneous occurrence of drought and pathogen during plant growth provokes complex pathways controlled by different signaling events resulting in positive or negative impact of one stress over the other. Here, we summarize the effect of combined drought and pathogen infection on plants and highlight the tailored strategies adapted by plants. Besides, we enumerate the evidences from pathogen derived elicitors and ABA response studies for understanding simultaneous drought and pathogen tolerance.

Plant defense responses at stomata and apoplast are the most important early events during plant-bacteria interactions. The key components for the signaling of stomatal defense and nonhost resistance have not been fully characterized. Here we report the newly identified small GTPase, Nucleolar GTP-binding protein 1 (NOG1), functions for plant immunity against bacterial pathogens. Virus-induced gene silencing of NOG1 compromised nonhost resistance in N. benthamiana and tomato. Comparative genomic analysis showed that two NOG1 copies are present in all known plant species: NOG1-1 and NOG1-2. Gene downregulation and overexpression studies of NOG1-1 and NOG1-2 in Arabidopsis revealed the novel function of these genes in nonhost resistance and stomatal defense against bacterial pathogens, respectively. Specially, NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimuli through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. The results here provide valuable information on the new functional role of small GTPase, NOG1, in guard cell signaling and early plant defense in response to bacterial pathogens.

Global transcriptome studies demonstrated the existence of unique plant responses under combined stress which are otherwise not seen during individual stresses. In order to combat combined stress plants use signaling pathways and ‘cross talk’ mediated by hormones involved in stress and growth related processes. However, interactions among hormones’ pathways in combined stressed plants are not yet known. Here we studied dynamics of different hormones under individual and combined drought and pathogen infection in Arabidopsis thaliana by liquid chromatography-mass spectrometry (LC-MS) based profiling. Our results revealed abscisic acid (ABA) and salicylic acid (SA) as key regulators under individual drought and pathogen stress respectively. Under combined drought and host pathogen stress (DH) we observed non-induced levels of ABA with an upsurge in SA and jasmonic acid (JA) concentrations, underscoring their role in basal tolerance against host pathogen. Under a non-host pathogen interaction with drought (DNH) stressed plants, ABA, SA and JA profiles were similar to those under DH or non-host pathogen alone. We propose that plants use SA/JA dependent signaling during DH stress which antagonize ABA biosynthesis and signaling pathways during early stage of stress. The study provides insights into hormone modulation at different time points during combined stress.

Chickpea (Cicer arietinum); the second largest legume grown worldwide is prone to drought and various pathogen infections. These drought and pathogen stresses often occur concurrently in the field conditions. However, the molecular events in response to that are largely unknown. The present study examines the transcriptome dynamics in chickpea plants exposed to a combination of water-deficit stress and Ralstonia solanacearum infection. R. solanacearum is a potential wilt disease causing pathogen in chickpea. Drought stressed chickpea plants were infected with this pathogen and the plants were allowed to experience progressive drought with 2 and 4 days of R. solanacearum infection called short duration stress (SD stresses) and long duration stress (LD stresses), respectively. Our study showed that R. solanacearum multiplication decreased under SD-combined stress compared to SD-pathogen but there was no significant change in LD-combined stress compared to LD-pathogen. The microarray analysis during these conditions showed that 821 and 1039 differentially expressed genes (DEGs) were unique to SD- and LD-combined stresses, respectively, when compared with individual stress conditions. Three and fifteen genes were common among all the SD-stress treatments and LD-stress treatments, respectively. Genes involved in secondary cell wall biosynthesis, alkaloid biosynthesis, defense related proteins, and osmo-protectants were up-regulated during combined stress. The expression of genes involved in lignin and cellulose biosynthesis were specifically up-regulated in SD-combined, LD-combined, and LD-pathogen stress. A close transcriptomic association of LD-pathogen stress with SD-combined stress was observed in this study which indicates that R. solanacearum infection also exerts drought stress along with pathogen stress thus mimics combined stress effect. Furthermore the expression profiling of candidate genes using real-time quantitative PCR validated the microarray data. The study showed that down-regulation of defense-related genes during LD-combined stress resulted in an increased bacterial multiplication as compared to SD-combined stress. Overall, our study highlights a sub-set of DEGs uniquely expressed in response to combined stress, which serve as potential candidates for further functional characterization to delineate the molecular response of the plant to concurrent drought-pathogen stress.

Plants under natural conditions encounter a number of abiotic and biotic stresses often being inflicted simultaneously. Plant responses to a stress are governed by intricate network of the hormone signaling pathways. Abscisic acid (ABA) forms the major component of the plant response to drought and cold stress. Salicylic acid (SA), jasmonic acid (JA), and ethylene act as key regulators of plant response to pathogen infection. In fact, the extensive cross talk among the different hormone-mediated signaling pathways determines plant response to a particular stress. A large number of studies focus on hormone signaling under individual drought and pathogen stresses and the cross talk between the two stress responses. However, owing to the relatively few studies on combined drought and pathogen stresses, our understanding of phytohormonal signaling under combined stress is still obscure. Recent studies on combined drought and pathogen infection indicate that plants when simultaneously exposed to the two stresses often exhibit a transcriptional and metabolic response different from that exhibited under single stress conditions. This is also applicable to the phytohormonal signaling. The nature, time, and severity of the two stresses in combination modulate hormonal concentrations as well as the hormone signal transduction pathways involved. In this chapter, we provide a compendious description of the role of the three major hormones, namely, ABA, SA, and JA, in combined drought and pathogen infection. A brief description of the role of auxins, cytokinins, and gibberellins has also been provided. Taking leads from few studies, we have discussed the potential role of hormones in conferring combined drought and pathogen stress tolerance to plants. We also briefly discussed the effect of different “stress elicitors” on hormone signaling.

Tissue water status plays an important role in determining the fate of plant-pathogen interaction. Water availability is one of the factors that determine the multiplication of bacteria on the surface and inside the plants. Plant-water relations are highly influenced by soil water status, and drought stress is known to severely impact plant-pathogen interaction. Water, as a limiting factor, is differentially manipulated by both plants and pathogens during compatible and incompatible interactions. Plants stimulate the localized loss of water at the site of infection for limiting the bacterial multiplication. On the other hand, foliar and vascular bacterial pathogens employ different strategies to alter the plant water status and eventually establish the infection in plants. Foliar pathogens manipulate their own machinery in response to water-limited condition in plants. They also modulate the plant machinery in order to promote disease by increasing the water soaking between the cells. Similarly, vascular pathogens use different strategies such as clogging of vessels and embolism of xylem elements that leads to wilting of plant. Here, we discuss the current knowledge on impact of drought stress during plant interaction with foliar or vascular pathogen interactions.

Global warming leads to the concurrence of a number of abiotic and biotic stresses, thus affecting agricultural productivity. Occurrence of abiotic stresses can alter plant–pest interactions by enhancing host plant susceptibility to pathogenic organisms, insects, and by reducing competitive ability with weeds. On the contrary, some pests may alter plant response to abiotic stress factors. Therefore, systematic studies are pivotal to understand the effect of concurrent abiotic and biotic stress conditions on crop productivity. However, to date, a collective database on the occurrence of various stress combinations in agriculturally prominent areas is not available. This review attempts to assemble published information on this topic, with a particular focus on the impact of combined drought and pathogen stresses on crop productivity. In doing so, this review highlights some agriculturally important morpho-physiological traits that can be utilized to identify genotypes with combined stress tolerance. In addition, this review outlines potential role of recent genomic tools in deciphering combined stress tolerance in plants. This review will, therefore, be helpful for agronomists and field pathologists in assessing the impact of the interactions between drought and plant-pathogens on crop performance. Further, the review will be helpful for physiologists and molecular biologists to design agronomically relevant strategies for the development of broad spectrum stress tolerant crops.

Nonhost resistance is a broad-spectrum plant defense that provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Upon landing on the surface of a nonhost plant species, a potential bacterial pathogen initially encounters preformed and, later, induced plant defenses. One of the initial defense responses from the plant is pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Nonhost plants also have mechanisms to detect nonhost-pathogen effectors and can trigger a defense response referred to as effector-triggered immunity (ETI). This nonhost resistance response often results in a hypersensitive response (HR) at the infection site. This review provides an overview of these plant defense strategies. We enumerate plant genes that impart nonhost resistance and the bacterial counter-defense strategies. In addition, prospects for application of nonhost resistance to achieve broad-spectrum and durable resistance in crop plants are also discussed.

In field conditions, plants are concurrently exposed to multiple stresses, where one stressor impacts the plant's response to another stressor, and the resultant net effect of these stresses differs from individual stress response. The present study investigated the effect of drought stress on interaction of chickpea with Pseudomonas syringae pv. phaseolicola (Psp; foliar pathogen) and Ralstonia solanacearum (Rs; xylem inhabiting wilt causing pathogen), respectively, and the net-effect of combined stress on chlorophyll content and cell death. Two type of stress treatments were used to study the influence of each stress factor during combined stress, viz., imposition of drought stress followed by pathogen challenge (DP), and pathogen inoculated plants imposed with drought in course of pathogen infection (PD). Drought stress was imposed at different levels with pathogen inoculum to understand the influence of different stress intensities on stress interaction and their net impact. Drought stressed chickpea plants challenged with Psp infection (DPsp) showed reduced in planta bacterial number compared to Psp infection alone. Similarly, Rs infection of chickpea plants showed reduced in planta bacterial number under severe drought stress. Combined drought and Psp (DPsp) infected plants showed decreased cell death compared to plants infected only with Psp but the extent of cell death was similar to drought stressed plants. Similarly, chlorophyll content in plants under combined stress was similar to the individual drought stressed plants; however, the chlorophyll content was more compared to pathogen only infected plants. Under combined drought and Rs infection (DRs), cell death was similar to individual drought stress but significantly less compared to only Rs infected plants. Altogether, the study proposes that both stress interaction and net effect of combined stress could be majorly influenced by first occurring stress, for example, drought stress in DP treatment. In addition, our results indicate that the outcome of the two stress interaction in plant depends on timing of stress occurrence and nature of infecting pathogen.

Plant responses to a combination of drought and bacterial pathogen infection, an agronomically important and altogether a new stress, are not well studied. While occurring concurrently, these two stresses can lead to synergistic or antagonistic effects on plants due to stress-interaction. It is reported that plant responses to the stress combinations consist of both strategies unique to combined stress and those shared between combined and individual stresses. However, the combined stress response mechanisms governing stress interaction and net impact are largely unknown. In order to study these adaptive strategies, an accurate and convenient methodology is lacking even in model plants like Arabidopsis thaliana. The gradual nature of drought stress imposition protocol poses a hindrance in simultaneously applying pathogen infection under laboratory conditions to achieve combined stress. In present study we aimed to establish systematic combined stress protocol and to study physiological responses of the plants to various degrees of combined stress. Here, we have comprehensively studied the impact of combined drought and Pseudomonas syringae pv. tomato DC3000 infection on A. thaliana. Further, by employing different permutations of drought and pathogen stress intensities, an attempt was made to dissect the contribution of each individual stress effects during their concurrence. We hereby present two main aspects of combined stress viz., stress interaction and net impact of the stress on plants. Mainly, this study establishes a systematic protocol to assess the impact of combined drought and bacterial pathogen stress. It was observed that as a result of net impact, some physiological responses under combined stress are tailored when compared to the plants exposed to individual stresses. We also infer that plant responses under combined stress in this study are predominantly influenced by the drought stress. Our results show that pathogen induced disease symptoms were ameliorated by drought stress in combined stressed plants. Combined stressed plants also displayed reduced ROS generation and declined cell death which could be attributed to activation of effective basal defence responses. We hypothesize a model on ABA mediated gene regulation to partly explain the possible mechanistic basis for reduced in planta bacterial numbers under combined stress over individual pathogen

With frequent fluctuations in global climate, plants are exposed to co-occurring drought and pathogen infection and this combination adversely affects plant survival. In the past, some studies indicated that morpho-physiological responses of plants to the combined stress are different from the individual stressed plants. However, interaction of drought stressed plants with pathogen has not been widely studied at molecular level. Such studies are important to understand the defense pathways that operate as part of combined stress tolerance mechanism. In this study, Arabidopsis thaliana was exposed to individual drought stress, Pseudomonas syringae pv tomato DC3000 (Pst DC3000) infection and their combination. Using Affymetrix WT gene 1.0 ST array, global transcriptome profiling of leaves under individual drought stress and pathogen infection was compared with their combination. The results obtained from pathway mapping (KAAS and MAPMAN) demonstrated the modulation in defense pathways in A. thaliana under drought and host pathogen Pst DC3000 infection. Further, our study revealed ‘tailored’ responses under combined stress and the time of occurrence of each stress during their concurrence has showed differences in transcriptome profile. Our results from microarray and RT-qPCR revealed unique regulation of 20 novel genes exclusively during the stress interaction. This study indicates that plants exposed to concurrent drought and pathogen stress experience a new state of stress. Thus, under frequently changing climatic conditions each combination of stressor and their timing defines the plant responses and should thus be studied explicitly.

Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised in nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens.

In nature plants are often simultaneously challenged by different biotic and abiotic stresses. Although the mechanisms underlying plant responses against single stress have been studied considerably, plant tolerance mechanisms under combined stress is not understood. Also, the mechanism used to combat independently and sequentially occurring many number of biotic and abiotic stresses has also not systematically studied. From this context, in this study, we attempted to explore the shared response of sunflower plants to many independent stresses by using meta-analysis of publically available transcriptome data and transcript profiling by quantitative PCR. Further, we have also analyzed the possible role of the genes so identified in contributing to combined stress tolerance. Meta-analysis of transcriptomic data from many abiotic and biotic stresses indicated the common representation of oxidative stress responsive genes. Further, menadione-mediated oxidative stress in sunflower seedlings showed similar pattern of changes in the oxidative stress related genes. Based on this a large scale screening of 55 sunflower genotypes was performed under menadione stress and those contrasting in oxidative stress tolerance were identified. Further to confirm the role of genes identified in individual and combined stress tolerance the contrasting genotypes were individually and simultaneously challenged with few abiotic and biotic stresses. The tolerant hybrid showed reduced levels of stress damage both under combined stress and few independent stresses. Transcript profiling of the genes identified from meta-analysis in the tolerant hybrid also indicated that the selected genes were up-regulated under individual and combined stresses. Our results indicate that menadione-based screening can identify genotypes not only tolerant to multiple number of individual biotic and abiotic stresses, but also the combined stresses.

Under field conditions plants are often challenged by combination of biotic and abiotic stressors and they severely affect crop productivity. An increasing number of studies suggest that plants “tailor” their adaptation strategies to combat simultaneously occurring stresses. The stress combat strategies of plants are customized according to the stress combination and vary with the intensity and timing of the stresses involved. While some of the responses seen under combined stress are commonly instigated by individual stresses, some other are uniquely triggered under combined stress. Since some responses are unique only to the combined stress, the outcome of a stress interaction cannot be completely predicted using results from individual stress studies. In this chapter, the effects of combinatorial drought stress and pathogen infection on plants are discussed with an emphasis on the molecular and physiological mechanisms that underpin how plants tolerate simultaneously occurring stresses. We also highlight the complexity involved in the responses of plants to multiple stresses and underscore the importance of studying plant stressors in combination.

Bacterial pathogens colonize a host plant by growing between the cells by utilizing the nutrients present in apoplastic space. While successful pathogens manipulate the plant cell membrane to retrieve more nutrients from the cell, the counteracting plant defense mechanism against nonhost pathogens to restrict the nutrient efflux into the apoplast is not clear. To identify the genes involved in nonhost resistance against bacterial pathogens, we developed a virus-induced gene-silencing-based fast-forward genetics screen in Nicotiana benthamiana. Silencing of N. benthamiana SQUALENE SYNTHASE, a key gene in phytosterol biosynthesis, not only compromised nonhost resistance to few pathovars of Pseudomonas syringae and Xanthomonas campestris, but also enhanced the growth of the host pathogen P. syringae pv tabaci by increasing nutrient efflux into the apoplast. An Arabidopsis (Arabidopsis thaliana) sterol methyltransferase mutant (sterol methyltransferase2) involved in sterol biosynthesis also compromised plant innate immunity against bacterial pathogens. The Arabidopsis cytochrome P450 CYP710A1, which encodes C22-sterol desaturase that converts β-sitosterol to stigmasterol, was dramatically induced upon inoculation with nonhost pathogens. An Arabidopsis Atcyp710A1 null mutant compromised both nonhost and basal resistance while overexpressors of AtCYP710A1 enhanced resistance to host pathogens. Our data implicate the involvement of sterols in plant innate immunity against bacterial infections by regulating nutrient efflux into the apoplast.

Virus-induced gene silencing (VIGS) is one of the commonly used RNA silencing methods in plant functional genomics. It is widely known that VIGS can occur for about 3 weeks. A few reports show that duration of VIGS can be prolonged for up to 3 months. Increasing the duration of endogenous gene silencing and developing a method for nonintegration-based persistent VIGS in progeny seedlings will widen the application of VIGS. We used three marker genes that provoke visible phenotypes in plants upon silencing to study persistence and transmittance of VIGS to progeny in two plant species, Nicotiana benthamiana and tomato. We used a Tobacco rattle virus (TRV)-based VIGS vector and showed that the duration of gene silencing by VIGS can occur for more than 2 years and that TRV is necessary for longer duration VIGS. Also, inoculation of TRV-VIGS constructs by both Agrodrench and leaf infiltration greatly increased the effectiveness and duration of VIGS. Our results also showed transmittance of VIGS to progeny seedlings via seeds. A longer silencing period will facilitate detailed study of target genes in plant development and stress tolerance. Further, the transmittance of VIGS to progeny will be useful in studying the effect of gene silencing in young seedlings. Our results provide a new dimension for the application of VIGS in plants.

Understanding post-transcriptional gene silencing (PTGS) phenomena in plants has provided breakthroughs in advancing plant functional genomics. A recently developed approach based on one of the strategies adopted by plants to defend against viruses, called virus-induced gene silencing (VIGS), is being widely used to enumerate the function of plant genes. Since its discovery, VIGS has been widely used to characterize plant genes involved in metabolic pathways, homeostasis, basic cellular functions, plant-microbe, plant-nematode and plant-herbivore interaction. Recently, the application of this technique has been extended to characterize the genes and cellular processes involved in abiotic-stress tolerance, and in particular drought and oxidative stress. Because abiotic-stress tolerance is multigenic, identification and characterization of genes involved in this process is challenging. VIGS could become one among the several potential tools in understanding the relevance of these stress-responsive genes. Development of VIGS protocols for the use of heterologous gene sequences as VIGS-inducers has extended its applicability to analyze genes of VIGS recalcitrant plant species. This article describes the methodology of VIGS for characterizing the water-deficit-stress-responsive genes, precautions to be taken during the experimentation, and future application of this technology as a fast forwarded as well as a reverse genetics tool to identify and characterize plant genes involved in drought tolerance. We also describe the importance of accurate water-deficit-stress imposition and quantification of stress-induced changes in the silenced plants during the process of screening to identify genes responsible for tolerance. Further, limitations of VIGS in characterizing the abiotic-stress-responsive genes are noted, with suggestions to overcome these limitations.