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Non-host resistance (NHR) is a prevalent mode of plant defense against invading pathogens, including viruses. Being hard to circumvent, NHR has considerable potential to provide durable resistance against virus infection. This review systematically analyzes recent research findings demonstrating NHR mechanisms and chronologically enumerates the defense strategies contributing to NHR. Conventionally, the NHR mechanisms that operate by restricting viral entry and multiplication are termed "true NHR." Here, we propose NHR mechanisms operating beyond viral multiplication in non-host plants as "apparent NHR." This review elucidates both categories of NHR and provides insights into the mechanisms involved in NHR. • Multiple layers of NHR mechanisms by plants operate against viruses. • NHR has immense potential to provide durable resistance against virus infection. • The current understanding about NHR is limited. • NHR mechanisms restricting viral entry and multiplication fall under "true NHR.". • Mechanisms arresting viral movement across cells, etc., are "apparent NHR.". [ABSTRACT FROM AUTHOR]

• Arabidopsis modulates the expression of ath-miR164c to regulate proline metabolism to adapt to the combined stress. • ath-miR164c regulates expression of AtP5CS1 gene. • Proline metabolism plays positive role under combined drought and Pseudomonas syringae pv. tomato DC3000 infection. 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. [ABSTRACT FROM AUTHOR]

• AtGBF3 is involved in combined drought and pathogen stress tolerance. • AtGBF3 is a common gene that plays a role under individual and combined stress. • AtGBF3 regulates combined stress tolerance by activating ABA-mediated signaling. • We developed a new method to apply PEG plus pathogen stress on MS-grown plants. 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. [ABSTRACT FROM AUTHOR]

Understanding the complex challenges that plants face from multiple stresses is key to developing climate-ready crops. We highlight the significance of the Stress Combinations and their Interactions in Plants database (SCIPdb) for studying the impact of stress combinations on plants and the importance of aligning thematic research programs to create crops aligned with achieving sustainable development goals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Drought dynamically influences the interactions between plants and pathogens, thereby affecting disease outbreaks. Understanding the intricate mechanistic aspects of the multiscale interactions among plants, pathogens, and the environment-known as the disease triangle-is paramount for enhancing the climate resilience of crop plants. In this review, we systematically compile and comprehensively analyse current knowledge on the influence of drought on the severity of plant diseases. We emphasise that studying these stresses in isolation is not sufficient to predict how plants respond to combined stress from both drought and pathogens. The impact of drought and pathogens on plants is complex and multifaceted, encompassing the activation of antagonistic signalling cascades in response to stress factors. The nature, intensity, and temporality of drought and pathogen stress occurrence significantly influence the outcome of diseases. We delineate the drought-sensitive nodes of plant immunity and highlight the emerging points of crosstalk between drought and defence signalling under combined stress. The limited mechanistic understanding of these interactions is acknowledged as a key research gap in this area. The information synthesised herein will be crucial for crafting strategies for the accurate prediction and mitigation of future crop disease risks, particularly in the context of a changing climate., (© 2024 John Wiley & Sons Ltd.)

Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba , a close relative of Brassicas, and the highly susceptible Brassica rapa . The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa . Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola , which will aid in devising strategies for breeding resistant varieties of oilseed Brassica., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ahmed, Dey, Senthil-Kumar, Modi, Sarmah and Bhorali.)

AtSWEET11 and AtSWEET12 are central players in phloem loading and long-distance sucrose translocation. During drought stress, these transporters enhance sucrose transport from shoot to root, increasing root proliferation. Chen et al. have now unravelled novel aspects of sucrose transport regulation, occurring via AtSWEET11 and AtSWEET12 phosphorylation and oligomerisation., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

With the increasing understanding of fundamentals of gene silencing pathways in plants, various tools and techniques for downregulating the expression of a target gene have been developed across multiple plant species. This chapter provides an insight into the molecular mechanisms of gene silencing and highlights the advancements in various gene silencing approaches. The prominent aspects of different gene silencing methods, their advantages and disadvantages have been discussed. A succinct discussion on the newly emerged microRNA-based technologies like microRNA-induced gene silencing (MIGS) and microRNA-mediated virus-induced gene silencing (MIR-VIGS) are also presented. We have also discussed the gene-editing system like CRISPR-Cas. The prominent bottlenecks in gene silencing methods are the off-target effects and lack of universal applicability. However, the tremendous growth in understanding of this field reflects the potentials for improvements in the currently available approaches and the development of new widely applicable methods for easy, fast, and efficient functional characterization of plant genes., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The high throughputness and affordability of "omics" technologies is leading to the identification of a large number of abiotic stress genes, with many of them responsive to multiple stresses. In vivo functional characterization of these genes under multiple stresses is challenging but essential to develop resilient crops for the changing climate. Here we describe a high-throughput Virus-Induced Gene Silencing-based methodology for functional analysis of genes under multiple abiotic stresses using leaf disks. Leaves with maximal silencing, which is localized to only a few leaves and to a short period, can be effectively used for multiple stress imposition and stress affect quantification., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., Competing Interests: Authors declare that they have no competing interest.

Understanding molecular mechanisms of transcriptional and posttranscriptional gene silencing pathways in plants over the past decades has led to development of tools and methods for silencing a target gene in various plant species. In this review chapter, both the recent understanding of molecular basis of gene silencing pathways and advances in various widely used gene silencing methods are compiled. We also discuss the salient features of the different methods like RNA interference (RNAi) and virus-induced gene silencing (VIGS) and highlight their advantages and disadvantages. Gene silencing technology is constantly progressing as reflected by rapidly emerging new methods. A succinct discussion on the recently developed methods like microRNA-mediated virus-induced gene silencing (MIR-VIGS) and microRNA-induced gene silencing (MIGS) is also provided. One major bottleneck in gene silencing approaches has been the associated off-target silencing. The other hurdle has been the lack of a universal approach that can be applied to all plants. For example, we face hurdles like incompatibility of VIGS vectors with the host and inability to use MIGS for plant species which are not easily transformable. However, the overwhelming research in this direction reflects the scope for overcoming the short comings of gene silencing technology.

Agroinoculation, first developed as a simple tool to study plant-virus interactions, is a popular method of choice for functional gene analysis of viral genomes. With the explosive growth of genomic information and the development of advanced vectors to dissect plant gene function, this reliable method of viral gene delivery in plants, has been recruited and morphed into a technique popularly known as agroinfiltration. This technique was developed to examine the effects of transient gene expression, with applications ranging from studies of plant-pathogen interactions, abiotic stresses, a variety of transient expression assays to study protein localization, and protein-protein interactions. We present a brief overview of literature which document both these applications, and then provide simple agroinoculation and agroinfiltration methods being used in our laboratory for functional gene analysis, as well as for fast-forward and reverse genetic screens using virus-induced gene silencing (VIGS).

RNA interference (RNAi), mediated by short interfering RNAs (siRNAs), is one of the widely used functional genomics method for suppressing the gene expression in plants. Initially, gene silencing by RNAi mechanism was believed to be specific requiring sequence homology between siRNA and target mRNA. However, several recent reports have showed that non-specific effects often referred as off-target gene silencing can occur during RNAi. This unintended gene silencing can lead to false conclusions in RNAi experiments that are aimed to study the functional role of a particular target gene in plants. This especially is a major problem in large-scale RNAi-based screens aiming for gene discovery. Hence, understanding the off-target effects is crucial for minimizing such effects to better conclude gene function analyzed by RNAi. We discuss here potential problems of off-target gene silencing and focus on possibilities that favor this effect during post-transcriptional gene silencing. Suggestions to overcome the off-target effects during RNAi studies are also presented. We believe that information available in present-day plant science literature about specificity of siRNA actions is inadequate. In-depth systematic studies to understand their molecular basis are necessary to enable improved design of more specific RNAi vectors. In the meantime, gene function and phenotype results from present-day RNAi studies need to be interpreted with caution.

In response to water-deficit stress, plants alter expression of thousands of genes and as a result, cellular, physiological, and biochemical processes are modified. Understanding the functional role of water-deficit stress-responsive genes is important in order to develop stress-tolerant plants. RNA interference (RNAi) technology is one of the potential reverse genetics tool for assessing the functional significance of these genes. We describe here the protocols for developing stable gene knockdown lines for stress-induced genes using RNAi. In addition, stress imposition method that allows plants to experience gradual acclimation stress is enumerated. Further, precautions that should be taken while developing RNAi lines and during stress imposition are discussed.