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This Protocol describes how to downregulate specific plant genes using tobacco rattle virus virus-induced gene silencing (TRV-VIGS). The method can be used in a range of plants, but N. benthamiana is used here as an example. 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.

Since 1999, various forward- and reverse-genetic approaches have uncovered nearly 200 genes required for symbiotic nitrogen fixation (SNF) in legumes. These discoveries advanced our understanding of the evolution of SNF in plants and its relationship to other beneficial endosymbioses, signaling between plants and microbes, the control of microbial infection of plant cells, the control of plant cell division leading to nodule development, autoregulation of nodulation, intracellular accommodation of bacteria, nodule oxygen homeostasis, the control of bacteroid differentiation, metabolism and transport supporting symbiosis, and the control of nodule senescence. This review catalogs and contextualizes all of the plant genes currently known to be required for SNF in two model legume species, Medicago truncatula and Lotus japonicus, and two crop species, Glycine max (soybean) and Phaseolus vulgaris (common bean). We also briefly consider the future of SNF genetics in the era of pan-genomics and genome editing.

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.

The MYB-basic helix–loop–helix (bHLH)-WD40 complexes regulating anthocyanin and proanthocyanidin (PA) biosynthesis in plants are not fully understood. Here Medicago truncatula bHLH MtTT8 was characterized as a central component of these ternary complexes that control anthocyanin and PA biosynthesis. Mttt8 mutant seeds have a transparent testa phenotype with reduced PAs and anthocyanins. MtTT8 restores PA and anthocyanin productions in Arabidopsis tt8 mutant. Ectopic expression of MtTT8 restores anthocyanins and PAs in mttt8 plant and hairy roots and further enhances both productions in wild-type hairy roots. Transcriptomic analyses and metabolite profiling of mttt8 mutant seeds and M. truncatula hairy roots (mttt8 mutant, mttt8 mutant complemented with MtTT8, or MtTT8 overexpression lines) indicate that MtTT8 regulates a subset of genes involved in PA and anthocyanin biosynthesis. MtTT8 is genetically regulated by MtLAP1, MtPAR and MtWD40-1. Combinations of MtPAR, MtLAP1, MtTT8 and MtWD40-1 activate MtTT8 promoter in yeast assay. MtTT8 interacts with these transcription factors to form regulatory complexes. MtTT8, MtWD40-1 and an MYB factor, MtPAR or MtLAP1, interacted and activated promoters of anthocyanidin reductase and anthocyanidin synthase to regulate PA and anthocyanin biosynthesis, respectively. Our results provide new insights into the complex regulation of PA and anthocyanin biosynthesis in M. truncatula.

Agrobacterium -mediated plant transformation (AMT) is the basis of modern-day plant biotechnology. One major drawback of this technology is the recalcitrance of many plant species/varieties to Agrobacterium infection, most likely caused by elicitation of plant defense responses. Here, we develop a strategy to increase AMT by engineering Agrobacterium tumefaciens to express a type III secretion system (T3SS) from Pseudomonas syringae and individually deliver the P. syringae effectors AvrPto, AvrPtoB, or HopAO1 to suppress host defense responses. Using the engineered Agrobacterium , we demonstrate increase in AMT of wheat, alfalfa and switchgrass by ~250%–400%. We also show that engineered A. tumefaciens expressing a T3SS can deliver a plant protein, histone H2A-1, to enhance AMT. This strategy is of great significance to both basic research and agricultural biotechnology for transient and stable transformation of recalcitrant plant species/varieties and to deliver proteins into plant cells in a non-transgenic manner. Agrobacterium infection can cause defense responses in many plants, which leads to transformation recalcitrance. Here, the authors express type III secretion system in Agrobacterium to deliver effector proteins into plant cells to suppress host defense responses and thus enhance transformation in some plant species.

Virus-induced gene silencing (VIGS) is an effective tool for gene function analysis in plants. Over the last decade, VIGS has been successfully used as both a forward and reverse genetics technique for gene function analysis in various model plants, as well as crop plants. With the increased identification of differentially expressed genes under various abiotic stresses through high-throughput transcript profiling, the application of VIGS is expected to be important in the future for functional characterization of a large number of genes. In the recent past, VIGS was proven to be an elegant tool for functional characterization of genes associated with abiotic stress responses. In this review, we provide an overview of how VIGS is used in different crop species to characterize genes associated with drought-, salt-, oxidative- and nutrient-deficiency-stresses. We describe the examples from studies where abiotic stress related genes are characterized using VIGS. In addition, we describe the major advantages of VIGS over other currently available functional genomics tools. We also summarize the recent improvements, limitations and future prospects of using VIGS as a tool for studying plant responses to abiotic stresses.

Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

Background Plant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member species Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 found to have beneficial traits including plant growth promotion and broad-spectrum antifungal activity towards taxonomically diverse plant pathogens. Understanding the genomes will assist in devising a bioformulation for crop protection while exploiting their beneficial functional roles. Results Two potential firmicute species were isolated from pearl millet panicles. Morphological, biochemical, and molecular characterization revealed their identities as Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36. The seed priming assays revealed the ability of both species to enhance plant growth promotion and seedling vigour index. Invitro assays with PBs 12 and PBl 36 showed the antibiosis effect against taxonomically diverse plant pathogens ( Magnaporthe grisea; Sclerotium rolfsii; Fusarium solani; Alternaria alternata; Ganoderma sp.) of crops and multipurpose tree species. The whole genome sequence analysis was performed to unveil the genetic potential of these bacteria for plant protection. The complete genomes of PBs 12 and PBl 36 consist of a single circular chromosome with a size of 4.02 and 4.33 Mb and 4,171 and 4,606 genes, with a G + C content of 43.68 and 45.83%, respectively. Comparative Average Nucleotide Identity (ANI) analysis revealed a close similarity of PBs 12 and PBl 36 with other beneficial strains of B. subtilis and B. paralicheniformis and found distant from B. altitudinis, B. amyloliquefaciens, and B. thuringiensis . Functional annotation revealed a majority of pathway classes of PBs 12 (30) and PBl 36 (29) involved in the biosynthesis of secondary metabolites, polyketides, and non-ribosomal peptides, followed by xenobiotic biodegradation and metabolism (21). Furthermore, 14 genomic regions of PBs 12 and 15 of PBl 36 associated with the synthesis of RiPP (Ribosomally synthesized and post-translationally modified peptides), terpenes, cyclic dipeptides (CDPs), type III polyketide synthases (T3PKSs), sactipeptides, lanthipeptides, siderophores, NRPS (Non-Ribosomal Peptide Synthetase), NRP-metallophone, etc. It was discovered that these areas contain between 25,458 and 33,000 secondary metabolite-coding MiBiG clusters which code for a wide range of products, such as antibiotics. The PCR-based screening for the presence of antimicrobial peptide (cyclic lipopeptide) genes in PBs 12 and 36 confirmed their broad-spectrum antifungal potential with the presence of spoVG, bacA, and srfAA AMP genes, which encode antimicrobial compounds such as subtilin, bacylisin, and surfactin. Conclusion The combined in vitro studies and genome analysis highlighted the antifungal potential of pearl millet panicle-associated Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36. The genetic ability to synthesize several antimicrobial compounds indicated the industrial value of PBs 12 and PBl 36, which shed light on further studies to establish their action as a biostimulant for crop protection.

Legumes develop symbiotic interactions with rhizobial bacteria to form nitrogen-fixing nodules. Bacterial Nod factors (NFs) and plant regulatory pathways modulating NF signalling control rhizobial infections and nodulation efficiency. Here we show that gibberellin (GA) signalling mediated by DELLA proteins inhibits rhizobial infections and controls the NF induction of the infection marker ENOD11 in Medicago truncatula . Ectopic expression of a constitutively active DELLA protein in the epidermis is sufficient to promote ENOD11 expression in the absence of symbiotic signals. We show using heterologous systems that DELLA proteins can interact with the nodulation signalling pathway 2 (NSP2) and nuclear factor-YA1 (NF-YA1) transcription factors that are essential for the activation of NF responses. Furthermore, MtDELLA1 can bind the ERN1 (ERF required for nodulation 1) promoter and positively transactivate its expression. Overall, we propose that GA-dependent action of DELLA proteins may directly regulate the NSP1/NSP2 and NF-YA1 activation of ERN1 transcription to regulate rhizobial infections. Bacterial Nod Factors (NF) regulate the formation of nitrogen fixing symbiotic nodules on legume roots. Here, the authors show that gibberellins act via DELLA proteins to regulate nodulation and propose that DELLA acts with NSP2 and NF-YA1 as a transcriptional co-activator of ERN1-dependent NF signalling.

The constant interactions between plants and pathogens in the environment and the resulting outcomes are of significant importance for agriculture and agricultural scientists. Disease resistance genes in plant cultivars can break down in the field due to the evolution of pathogens under high selection pressure. Thus, the protection of crop plants against pathogens is a continuous arms race. Like any other type of crop plant, legumes are susceptible to many pathogens. The dawn of the genomic era, in which high-throughput and cost-effective genomic tools have become available, has revolutionized our understanding of the complex interactions between legumes and pathogens. Genomic tools have enabled a global view of transcriptome changes during these interactions, from which several key players in both the resistant and susceptible interactions have been identified. This review summarizes some of the large-scale genomic studies that have clarified the host transcriptional changes during interactions between legumes and their plant pathogens while highlighting some of the molecular breeding tools that are available to introgress the traits into breeding programs. These studies provide valuable insights into the molecular basis of different levels of host defenses in resistant and susceptible interactions.