RAV1 family members function as transcriptional regulators and play a positive role in plant disease resistance.

Phytopathogens pose a severe threat to agriculture and strengthening the plant defense response is an important strategy for disease control. Here, we report that AtRAV1, an AP2 and B3 domain-containing transcription factor, is required for basal plant defense in Arabidopsis thaliana. The atrav1 mutant lines demonstrate hyper-susceptibility against fungal pathogens (Rhizoctonia solani and Botrytis cinerea), whereas AtRAV1 overexpressing lines exhibit disease resistance against them. Enhanced expression of various defense genes and activation of mitogen-activated protein kinases (AtMPK3 and AtMPK6) are observed in the R. solani infected overexpressing lines, but not in the atrav1 mutant plants. An in vitro phosphorylation assay suggests AtRAV1 to be a novel phosphorylation target of AtMPK3. Bimolecular fluorescence complementation and yeast two-hybrid assays support physical interactions between AtRAV1 and AtMPK3. Overexpression of the native as well as phospho-mimic but not the phospho-defective variant of AtRAV1 imparts disease resistance in the atrav1 mutant A. thaliana lines. On the other hand, overexpression of AtRAV1 fails to impart disease resistance in the atmpk3 mutant. These analyses emphasize that AtMPK3-mediated phosphorylation of AtRAV1 is important for the elaboration of the defense response in A. thaliana. Considering that RAV1 homologs are conserved in diverse plant species, we propose that they can be gainfully deployed to impart disease resistance in agriculturally important crop plants. Indeed, overexpression of SlRAV1 (a member of the RAV1 family) imparts disease tolerance against not only fungal (R. solani and B. cinerea), but also against bacterial (Ralstonia solanacearum) pathogens in tomato, whereas silencing of the gene enhances disease susceptibility.

A prophage tail-like protein facilitates the endophytic growth of Burkholderia gladioli and mounting immunity in tomato.

A prophage tail-like protein (Bg_9562) of Burkholderia gladioli strain NGJ1 possesses broad-spectrum antifungal activity, and it is required for the bacterial ability to forage over fungi. Here, we analyzed whether heterologous overexpression of Bg_9562 or exogenous treatment with purified protein can impart disease tolerance in tomato. The physiological relevance of Bg_9562 during endophytic growth of NGJ1 was also investigated. Bg_9562 overexpressing lines demonstrate fungal and bacterial disease tolerance. They exhibit enhanced expression of defense genes and activation of mitogen-activated protein kinases. Treatment with Bg_9562 protein induces defense responses and imparts immunity in wild-type tomato. The defense-inducing ability lies within 18-51 aa region of Bg_9562 and is due to sequence homology with the bacterial flagellin epitope. Interaction studies suggest that Bg_9562 is perceived by FLAGELLIN-SENSING 2 homologs in tomato. The silencing of SlSERK3s (BAK1 homologs) prevents Bg_9562-triggered immunity. Moreover, type III secretion system-dependent translocation of Bg_9562 into host apoplast is important for elicitation of immune responses during colonization of NGJ1. Our study emphasizes that Bg_9562 is important for the endophytic growth of B. gladioli, while the plant perceives it as an indirect indicator of the presence of bacteria to mount immune responses. The findings have practical implications for controlling plant diseases.

Concurrent overexpression of rice G-protein ß and γ subunits provide enhanced tolerance to sheath blight disease and abiotic stress in rice.

MAIN CONCLUSION: Our study demonstrates that simultaneous overexpression of RGB1 and RGG1 genes provides multiple stress tolerance in rice by inducing stress responsive genes and better management of ROS scavenging/photosynthetic machineries. The heterotrimeric G-proteins act as signalling molecules and modulate various cellular responses including stress tolerance in eukaryotes. The gamma (γ) subunit of rice G-protein (RGG1) was earlier reported to promote salinity stress tolerance in rice. In the present study, we report that a rice gene-encoding beta (ß) subunit of G-protein (RGB1) gets upregulated during both biotic (upon a necrotrophic fungal pathogen, Rhizoctonia solani infection) and drought stresses. Marker-free transgenic IR64 rice lines that simultaneously overexpress both RGB1 and RGG1 genes under CaMV35S promoter were raised. The overexpressing (OE) lines showed enhanced tolerance to R. solani infection and salinity/drought stresses. Several defense marker genes including OsMPK3 were significantly upregulated in the R. solani-infected OE lines. We also found the antioxidant machineries to be upregulated during salinity as well as drought stress in the OE lines. Overall, the present study provides evidence that concurrent overexpression of G-protein subunits (RGG1 and RGB1) impart multiple (both biotic and abiotic) stress tolerance in rice which could be due to the enhanced expression of stress-marker genes and better management of reactive oxygen species (ROS)-scavenging/photosynthetic machinery. The current study suggests an improved approach for simultaneous improvement of biotic and abiotic stress tolerance in rice which remains a major challenge for its sustainable cultivation.

Function of heterotrimeric G-protein γ subunit RGG1 in providing salinity stress tolerance in rice by elevating detoxification of ROS.

MAIN CONCLUSION: The present study provides evidence of a unique function of RGG1 in providing salinity stress tolerance in transgenic rice without affecting yield. It also provides a good example for signal transduction from the external environment to inside for enhanced agricultural production that withstands the extreme climatic conditions and ensures food security. The role of heterotrimeric G-proteins functioning as signalling molecules has not been studied as extensively in plants as in animals. Recently, their importance in plant stress signalling has been emerging. In this study, the function of rice G-protein γ subunit (RGG1) in the promotion of salinity tolerance in rice (Oryza sativa L. cv. IR64) was investigated. The overexpression of RGG1 driven by the CaMV35S promoter in transgenic rice conferred high salinity tolerance even in the presence of 200 mM NaCl. Transcript levels of antioxidative genes, i.e., CAT, APX, and GR, and their enzyme activities increased in salinity-stressed transgenic rice plants suggesting a better antioxidant system to cope the oxidative-damages caused by salinity stress. The RGG1-induced signalling events that conferred tolerance to salinity was mediated by increased gene expression of the enzymes that scavenged reactive oxygen species. In salinity-stressed RGG1 transgenic lines, the transcript levels of RGG2, RGB, RGA, DEP1, and GS3 also increased in addition to RGG1. These observations suggest that most likely the stoichiometry of the G-protein complex was not disturbed under stress. Agronomic parameters, endogenous sugar content (glucose and fructose) and hormones (GA3, zeatin and IAA) were also higher in the transgenic plants compared with the wild-type plants. A BiFC assay confirmed the interaction of RGG1 with different stress-responsive proteins which play active roles in signalling and prevention of aggregation of proteins under stress-induced perturbation. The present study will help in understanding the G-protein-mediated stress tolerance in plants.

CRISPR/Cas-Mediated Genome Engineering in Plants: Application and Prospectives.

Genetic engineering has become an essential element in developing climate-resilient crops and environmentally sustainable solutions to respond to the increasing need for global food security. Genome editing using CRISPR/Cas [Clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas)] technology is being applied to a variety of organisms, including plants. This technique has become popular because of its high specificity, effectiveness, and low production cost. Therefore, this technology has the potential to revolutionize agriculture and contribute to global food security. Over the past few years, increasing efforts have been seen in its application in developing higher-yielding, nutrition-rich, disease-resistant, and stress-tolerant "crops", fruits, and vegetables. Cas proteins such as Cas9, Cas12, Cas13, and Cas14, among others, have distinct architectures and have been used to create new genetic tools that improve features that are important for agriculture. The versatility of Cas has accelerated genomic analysis and facilitated the use of CRISPR/Cas to manipulate and alter nucleic acid sequences in cells of different organisms. This review provides the evolution of CRISPR technology exploring its mechanisms and contrasting it with traditional breeding and transgenic approaches to improve different aspects of stress tolerance. We have also discussed the CRISPR/Cas system and explored three Cas proteins that are currently known to exist: Cas12, Cas13, and Cas14 and their potential to generate foreign-DNA-free or non-transgenic crops that could be easily regulated for commercialization in most countries.

Heterologous overexpression of PDH45 gene of pea provides tolerance against sheath blight disease and drought stress in rice.

Biotic and abiotic stress tolerant crops are required for sustainable agriculture as well as ensuring global food security. In a previous study, we have reported that heterologous overexpression of pea DNA helicase (PDH45), a DEAD-box family member protein, provides salinity stress tolerance in rice. The improved management of photosynthetic machinery and scavenging of reactive oxygen species (ROS) are associated with PDH45 mediated salinity stress tolerance. However, the role of PDH45 in biotic and other abiotic stress (drought) tolerance remains unexplored. In the present study, we have generated marker-free transgenic IR64 rice lines that overexpress PDH45 under the CaMV35S promoter. The transgenic rice lines exhibited a significant level of tolerance against sheath blight disease, caused by Rhizoctonia solani, a polyphagous necrotrophic fungal pathogen. The defense as well as antioxidant responsive marker genes were significantly upregulated in the PDH45 overexpressing (OE) rice lines, upon pathogen infection. Moreover, the OE lines exhibited tolerance to drought stress and various antioxidant as well as drought responsive marker genes were significantly upregulated in them, upon drought stress. Overall, the current study emphasizes that heterologous overexpression of PDH45 provides abiotic as well as biotic stress tolerance in rice. Tolerance against drought as well as sheath blight disease by overexpression of a single gene (PDH45) signifies the practical implication of the present study. Moreover, considering the conserved nature of the gene in different plant species, we anticipate that PDH45 can be gainfully deployed to impart tolerance against multiple stresses in agriculturally important crops.

Proteomic and Genomic Studies of Micronutrient Deficiency and Toxicity in Plants.

Micronutrients are essential for plants. Their growth, productivity and reproduction are directly influenced by the supply of micronutrients. Currently, there are eight trace elements considered to be essential for higher plants: Fe, Zn, Mn, Cu, Ni, B, Mo, and Cl. Possibly, other essential elements could be discovered because of recent advances in nutrient solution culture techniques and in the commercial availability of highly sensitive analytical instrumentation for elemental analysis. Much remains to be learned about the physiology of micronutrient absorption, translocation and deposition in plants, and about the functions they perform in plant growth and development. With the recent advancements in the proteomic and molecular biology tools, researchers have attempted to explore and address some of these questions. In this review, we summarize the current knowledge of micronutrients in plants and the proteomic/genomic approaches used to study plant nutrient deficiency and toxicity.

Silencing of tomato CTR1 provides enhanced tolerance against Tomato leaf curl virus infection.

Tomato leaf curl virus (ToLCV) belonging to Begomovirus family of Geminivirus is known to cause one of the most destructive diseases in tomato. Amongst various ToLCVs, a monopartite Tomato leaf curl Joydebpur virus (ToLCJoV) is most prevalent in eastern part of India. In the present study, we observed induced expression of one of the negative regulators of ethylene signaling pathway gene (LeCTR1) in ToLCJoV infected plants. The Tobacco rattle virus (TRV) induced silencing of the LeCTR1 gene provided enhanced tolerance to ToLCJoV infections. The leaf curling as well as ROS accumulation was significantly reduced in the viral infected LeCTR1 silenced plants. Induction of several defense marker genes (NPR1, PR1, PR5, AOS2, EIN2, EIN3 and ERF5) reinforced enhanced tolerance against ToLCJoV infection in the LeCTR1 silenced tomato. Overall, the present study provides evidence that silencing of LeCTR1 can be deployed to protect tomato from ToLCJoV infections.

Bacteria-fungal Confrontation and Fungal Growth Prevention Assay.

There are some bacteria which can grow and multiply at the cost of living fungal biomass. They can potentially utilize fungi as a source of nutrients to forage over them. Such phenomenon is known as bacterial mycophagy, however, its mechanistic insights need to be explored to identify the molecules involved in mycophagy for potential utilization in controlling various fungal diseases. Recently we have demonstrated that a rice-associated bacteria Burkholderia gladioli strain NGJ1 exhibits mycophagous ability on several fungi, including Rhizoctonia solani, the necrotrophic fungal pathogen causing sheath blight disease in rice. We hereby describe our validated and efficient methods used to study B. gladioli strain NGJ1-R. solani interactions. These methodologies would be useful for designing assays to study the confrontation between bacteria and fungi which in turn enable discovery of novel antifungal molecules from such bacteria.

A prophage tail-like protein is deployed by Burkholderia bacteria to feed on fungi.

Some bacteria can feed on fungi, a phenomenon known as mycophagy. Here we show that a prophage tail-like protein (Bg_9562) is essential for mycophagy in Burkholderia gladioli strain NGJ1. The purified protein causes hyphal disintegration and inhibits growth of several fungal species. Disruption of the Bg_9562 gene abolishes mycophagy. Bg_9562 is a potential effector secreted by a type III secretion system (T3SS) and is translocated into fungal mycelia during confrontation. Heterologous expression of Bg_9562 in another bacterial species, Ralstonia solanacearum, confers mycophagous ability in a T3SS-dependent manner. We propose that the ability to feed on fungi conferred by Bg_9562 may help the bacteria to survive in certain ecological niches. Furthermore, considering its broad-spectrum antifungal activity, the protein may be potentially useful in biotechnological applications to control fungal diseases.Some bacteria can feed on live fungi through unclear mechanisms. Here, the authors show that a T3SS-secreted protein, which is homologous to phage tail proteins, allows a Burkholderia gladioli strain to kill and feed on various fungal species.