Muthappa Senthil-Kumar

Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense

Plants are constantly exposed to microorganisms in the environment and, as a result, have evolved intricate mechanisms to recognize and defend themselves against potential pathogens. One of these responses is the downregulation of photosynthesis and other processes associated with primary metabolism that are essential for plant growth. It has been suggested that the energy saved by downregulation of primary metabolism is diverted and used for defense responses. However, several studies have shown that upregulation of primary metabolism also occurs during plant-pathogen interactions. We propose that upregulation of primary metabolism modulates signal transduction cascades that lead to plant defense responses. In support of this thought, we here compile evidence from the literature to show that upon exposure to pathogens or elicitors, plants induce several genes associated with primary metabolic pathways, such as those involved in the synthesis or degradation of carbohydrates, amino acids and lipids. In addition, genetic studies have confirmed the involvement of these metabolic pathways in plant defense responses. This review provides a new perspective highlighting the relevance of primary metabolism in regulating plant defense against pathogens with the hope to stimulate further research in this area.

New dimensions for VIGS in plant functional genomics

Virus-induced gene silencing (VIGS) is an efficient tool for gene function studies. It has been used to perform both forward and reverse genetics to identify plant genes involved in several plant processes. However, this technology has not yet been used to its full potential. This can be attributed to several of its limitations such as inability to silence genes during seed germination and the non-stable nature of silencing. However, several recent studies have shown that these limitations can now be overcome. In this review, we will discuss the limitations of VIGS and suitable solutions. In addition, we also describe the recent improvements and future prospects of using VIGS in plant biology.

Glycolate Oxidase Modulates Reactive Oxygen Species–Mediated Signal Transduction during Nonhost Resistance in Nicotiana benthamiana and Arabidopsis

This article identifies the peroxisomal enzyme glycolate oxidase (GOX) as an essential element of nonhost resistance and provides evidence that apart from NADPH oxidase–generated H2O2, GOX is an alternate source for H2O2 production and plays a crucial role during both gene-for-gene and nonhost resistance by regulating genes from different defense signal transduction cascades. In contrast to gene-for-gene disease resistance, nonhost resistance governs defense responses to a broad range of potential pathogen species. To identify specific genes involved in the signal transduction cascade associated with nonhost disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana, and identified the peroxisomal enzyme glycolate oxidase (GOX) as an essential component of nonhost resistance. GOX-silenced N. benthamiana and Arabidopsis thaliana GOX T-DNA insertion mutants are compromised for nonhost resistance. Moreover, Arabidopsis gox mutants have lower H2O2 accumulation, reduced callose deposition, and reduced electrolyte leakage upon inoculation with hypersensitive response–causing nonhost pathogens. Arabidopsis gox mutants were not affected in NADPH oxidase activity, and silencing of a gene encoding NADPH oxidase (Respiratory burst oxidase homolog) in the gox mutants did not further increase susceptibility to nonhost pathogens, suggesting that GOX functions independently from NADPH oxidase. In the two gox mutants examined (haox2 and gox3), the expression of several defense-related genes upon nonhost pathogen inoculation was decreased compared with wild-type plants. Here we show that GOX is an alternative source for the production of H2O2 during both gene-for-gene and nonhost resistance responses.

The interactive effects of simultaneous biotic and abiotic stresses on plants: Mechanistic understanding from drought and pathogen combination

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.

Nonhost Resistance Against Bacterial Pathogens: Retrospectives and Prospects

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.

Shared and unique responses of plants to multiple individual stresses and stress combinations: physiological and molecular mechanisms.

In field conditions, plants are often simultaneously exposed to multiple biotic and abiotic stresses resulting in substantial yield loss. Plants have evolved various physiological and molecular adaptations to protect themselves under stress combinations. Emerging evidences suggest that plant responses to a combination of stresses are unique from individual stress responses. In addition, plants exhibit shared responses which are common to individual stresses and stress combination. In this review, we provide an update on the current understanding of both unique and shared responses. Specific focus of this review is on heat–drought stress as a major abiotic stress combination and, drought–pathogen and heat–pathogen as examples of abiotic–biotic stress combinations. We also comprehend the current understanding of molecular mechanisms of cross talk in relation to shared and unique molecular responses for plant survival under stress combinations. Thus, the knowledge of shared responses of plants from individual stress studies and stress combinations can be utilized to develop varieties with broad spectrum stress tolerance.

Phytosterols Play a Key Role in Plant Innate Immunity against Bacterial Pathogens by Regulating Nutrient Efflux into the Apoplast

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.

Tobacco rattle virus–based virus-induced gene silencing in Nicotiana benthamiana

Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) is widely used in various plant species to downregulate the expression of a target plant gene. TRV is a bipartite, positive-strand RNA virus with the TRV1 and TRV2 genomes. To induce post-transcriptional gene silencing (PTGS), the TRV2 genome is genetically modified to carry a fragment of the target gene and delivered into the plant (along with the TRV1 genome) by agroinoculation. TRV1- and TRV2-carrying Agrobacterium strains are then co-inoculated into 3-week-old plant leaves by one of three methods: a needleless syringe, the agrodrench method or by pricking with a toothpick. Target gene silencing occurs in the newly developed noninoculated leaves within 2–3 weeks of TRV inoculation. The TRV-VIGS protocol described here takes only 4 weeks to implement, and it is faster and easier to perform than other gene silencing techniques that are currently available. Although we use Nicotiana benthamiana as an example, the protocol is adaptable to other plant species.

Ornithine‐delta‐aminotransferase and proline dehydrogenase genes play a role in non‐host disease resistance by regulating pyrroline‐5‐carboxylate metabolism‐induced hypersensitive response

Non-host disease resistance involves the production of hypersensitive response (HR), a programmed cell death (PCD) that occurs at the site of pathogen infection. Plant mitochondrial reactive oxygen species (ROS) production and red-ox changes play a major role in regulating such cell death. Proline catabolism reactions, especially pyrroline-5-carboxylate (P5C) accumulation, are known to produce ROS and contribute to cell death. Here we studied important genes related to proline synthesis and catabolism in the defence against host and non-host strains of Pseudomonas syringae in Nicotiana benthamiana and Arabidopsis. Our results show that ornithine delta-aminotransferase (δOAT) and proline dehydrogenases (ProDH1 and ProDH2) are involved in the defence against non-host pathogens. Silencing of these genes in N. benthamiana delayed occurrence of HR and favoured non-host pathogen growth. Arabidopsis mutants for these genes compromised non-host resistance and showed a decrease in non-host pathogen-induced ROS. Some of the genes involved in proline metabolism were also induced by a pathogen-carrying avirulence gene, indicating that proline metabolism is influenced during effector-triggered immunity (ETI). Our results demonstrate that δOAT and ProDH enzyme-mediated steps produce ROS in mitochondria and regulate non-host HR, thus contributing to non-host resistance in plants.

Virus‐induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato

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.