RNAi, a sword of plant seeds to combat viral infections.

Vertical transmission of plant viruses through seeds has been known for a century, yet the mechanism for seeds to combat viral infection remains unclear. In this issue of Cell Host & Microbe, Liu and Ding demonstrate the genetic requirement of RNA silencing (RNAi) pathway for plants to suppress seed transmission.

Plant Immunity against Tobamoviruses.

Tobamoviruses are a group of plant viruses that pose a significant threat to agricultural crops worldwide. In this review, we focus on plant immunity against tobamoviruses, including pattern-triggered immunity (PTI), effector-triggered immunity (ETI), the RNA-targeting pathway, phytohormones, reactive oxygen species (ROS), and autophagy. Further, we highlight the genetic resources for resistance against tobamoviruses in plant breeding and discuss future directions on plant protection against tobamoviruses.

The NLR immune receptor ADR1 and lipase-like proteins EDS1 and PAD4 mediate stomatal immunity in Nicotiana benthamiana and Arabidopsis.

In the presence of pathogenic bacteria, plants close their stomata to prevent pathogen entry. Intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogenic effectors and activate effector-triggered immune responses. However, the regulatory and molecular mechanisms of stomatal immunity involving NLR immune receptors are unknown. Here, we show that the Nicotiana benthamiana RPW8-NLR central immune receptor ACTIVATED DISEASE RESISTANCE 1 (NbADR1), together with the key immune proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (NbEDS1) and PHYTOALEXIN DEFICIENT 4 (NbPAD4), plays an essential role in bacterial pathogen- and flg22-induced stomatal immunity by regulating the expression of salicylic acid (SA) and abscisic acid (ABA) biosynthesis or response-related genes. NbADR1 recruits NbEDS1 and NbPAD4 in stomata to form a stomatal immune response complex. The transcription factor NbWRKY40e, in association with NbEDS1 and NbPAD4, modulates the expression of SA and ABA biosynthesis or response-related genes to influence stomatal immunity. NbADR1, NbEDS1, and NbPAD4 are required for the pathogen infection-enhanced binding of NbWRKY40e to the ISOCHORISMATE SYNTHASE 1 promoter. Moreover, the ADR1-EDS1-PAD4 module regulates stomatal immunity in Arabidopsis (Arabidopsis thaliana). Collectively, our findings show the pivotal role of the core intracellular immune receptor module ADR1-EDS1-PAD4 in stomatal immunity, which enables plants to limit pathogen entry.

Plant virology in the 21st century in China: Recent advances and future directions.

Plant viruses are a group of intracellular pathogens that persistently threaten global food security. Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years, including basic research and technologies for preventing and controlling plant viral diseases. Here, we review these milestones and advances, including the identification of new crop-infecting viruses, dissection of pathogenic mechanisms of multiple viruses, examination of multilayered interactions among viruses, their host plants, and virus-transmitting arthropod vectors, and in-depth interrogation of plant-encoded resistance and susceptibility determinants. Notably, various plant virus-based vectors have also been successfully developed for gene function studies and target gene expression in plants. We also recommend future plant virology studies in China.

Plant airborne defense against insects, viruses, and beyond.

Plants emit volatiles as signals to trigger broad physiological responses, including airborne defense (AD). Gong et al. (Nature 2023; 622: 139-145) recently reported the genetic framework of how plants use AD to combat aphids and viruses. The study elucidates the mutualistic relationships between aphids and the viruses they transmit, revealing the broad biological and ecological significance of AD.

Molecular basis of methyl-salicylate-mediated plant airborne defence.

Aphids transmit viruses and are destructive crop pests1. Plants that have been attacked by aphids release volatile compounds to elicit airborne defence (AD) in neighbouring plants2-5. However, the mechanism underlying AD is unclear. Here we reveal that methyl-salicylate (MeSA), salicylic acid-binding protein-2 (SABP2), the transcription factor NAC2 and salicylic acid-carboxylmethyltransferase-1 (SAMT1) form a signalling circuit to mediate AD against aphids and viruses. Airborne MeSA is perceived and converted into salicylic acid by SABP2 in neighbouring plants. Salicylic acid then causes a signal transduction cascade to activate the NAC2-SAMT1 module for MeSA biosynthesis to induce plant anti-aphid immunity and reduce virus transmission. To counteract this, some aphid-transmitted viruses encode helicase-containing proteins to suppress AD by interacting with NAC2 to subcellularly relocalize and destabilize NAC2. As a consequence, plants become less repellent to aphids, and more suitable for aphid survival, infestation and viral transmission. Our findings uncover the mechanistic basis of AD and an aphid-virus co-evolutionary mutualism, demonstrating AD as a potential bioinspired strategy to control aphids and viruses.

Cotton leaf curl Multan virus C4 protein suppresses autophagy to facilitate viral infection.

Autophagy plays an important role in plant antiviral defense. Several plant viruses are reported to encode viral suppressor of autophagy (VSA) to prevent autophagy for effective virus infection. However, whether and how other viruses, in particular DNA viruses, also encode VSAs to affect viral infection in plants is unknown. Here, we report that the C4 protein encoded by Cotton leaf curl Multan geminivirus (CLCuMuV) inhibits autophagy by binding to the autophagy negative regulator eukaryotic translation initiation factor 4A (eIF4A) to enhance the eIF4A-Autophagy-related protein 5 (ATG5) interaction. By contrast, the R54A or R54K mutation in C4 abolishes its capacity to interact with eIF4A, and neither C4R54A nor C4R54K can suppress autophagy. However, the R54 residue is not essential for C4 to interfere with transcriptional gene silencing or post-transcriptional gene silencing. Moreover, plants infected with mutated CLCuMuV-C4R54K develop less severe symptoms with decreased levels of viral DNA. These findings reveal a molecular mechanism underlying how the DNA virus CLCuMuV deploys a VSA to subdue host cellular antiviral autophagy defense and uphold viral infection in plants.

Plant Defense and Viral Counter-Defense during Plant-Geminivirus Interactions.

Geminiviruses are the largest family of plant viruses that cause severe diseases and devastating yield losses of economically important crops worldwide. In response to geminivirus infection, plants have evolved ingenious defense mechanisms to diminish or eliminate invading viral pathogens. However, increasing evidence shows that geminiviruses can interfere with plant defense response and create a suitable cell environment by hijacking host plant machinery to achieve successful infections. In this review, we discuss recent findings about plant defense and viral counter-defense during plant-geminivirus interactions.

Southern rice black-streaked dwarf virus induces incomplete autophagy for persistence in gut epithelial cells of its vector insect.

Autophagy plays an important role in virus infection of the host, because viral components and particles can be degraded by the host's autophagy and some viruses may be able to hijack and subvert autophagy for its benefit. However, details on the mechanisms that govern autophagy for immunity against viral infections or benefit viral survival remain largely unknown. Plant reoviruses such as southern rice black-streaked dwarf virus (SRBSDV), which seriously threaten crop yield, are only transmitted by vector insects. Here, we report a novel mechanism by which SRBSDV induces incomplete autophagy by blocking autophagosome-lysosome fusion, resulting in viral accumulation in gut epithelial cells of its vector, white-backed planthopper (Sogatella furcifera). SRBSDV infection leads to stimulation of the c-Jun N-terminal kinase (JNK) signaling pathway, which further activates autophagy. Mature and assembling virions were found close to the edge7 of the outer membrane of autophagosomes. Inhibition autophagy leads to the decrease of autophagosomes, which resulting in impaired maturation of virions and the decrease of virus titer, whereas activation of autophagy facilitated virus titer. Further, SRBSDV inhibited fusion of autophagosomes and lysosomes by interacting with lysosomal-associated membrane protein 1 (LAMP1) using viral P10. Thus, SRBSDV not only avoids being degrading by lysosomes, but also further hijacks these non-fusing autophagosomes for its subsistence. Our findings reveal a novel mechanism of reovirus persistence, which can explain why SRBSDV can be acquired and transmitted rapidly by its insect vector.

Plant UVRAG interacts with ATG14 to regulate autophagosome maturation and geminivirus infection.

Autophagy is an essential degradation pathway that assists eukaryote survival under multiple stress conditions. Autophagosomes engulfing cargoes accomplish degradation only when they have matured through fusing with lysosomes or vacuoles. However, the molecular machinery mediating autophagosome maturation in plants remains unknown. Using the combined approaches of mass spectrometry, biochemistry, reverse genetics and microscopy, we uncover that UVRAG, a subunit of the class III phosphatidylinositol 3-kinase complexes in Nicotiana benthamiana, plays an essential role in autophagsome maturation via ATG14-assisted recruitment to autophagosomes and by facilitating RAB7 activation. An interaction between N. benthamiana UVRAG and ATG14 was observed in vitro and in vivo, which strikingly differed from their mutually exclusive appearance in different PI3KC3 complexes in yeast and mammals. This interaction increased the localisation of UVRAG on autophagosomes and enabled the convergence of autophagic and late endosomal structures, where they contributed to fusions between these two types of organelles by recruiting the essential membrane fusion factors RAB7 GTPase and the homotypic fusion and protein sorting (HOPS) complex. In addition, we uncovered a joint contribution of ATG14 and UVRAG to geminiviral infection, beyond autophagy. Our study provides insights into the mechanisms of autophagosome maturation in plants and expands the understanding of organisations and roles of the PI3KC3 complexes.

Linking Autophagy to Potential Agronomic Trait Improvement in Crops.

Autophagy is an evolutionarily conserved catabolic process in eukaryotic cells, by which the superfluous or damaged cytoplasmic components can be delivered into vacuoles or lysosomes for degradation and recycling. Two decades of autophagy research in plants uncovers the important roles of autophagy during diverse biological processes, including development, metabolism, and various stress responses. Additionally, molecular machineries contributing to plant autophagy onset and regulation have also gradually come into people's sights. With the advancement of our knowledge of autophagy from model plants, autophagy research has expanded to include crops in recent years, for a better understanding of autophagy engagement in crop biology and its potentials in improving agricultural performance. In this review, we summarize the current research progress of autophagy in crops and discuss the autophagy-related approaches for potential agronomic trait improvement in crop plants.

Autophagy in plant viral infection.

Autophagy is a conserved degradation pathway that delivers dysfunctional cellular organelles or other cytosol components to degradative vesicular structures (vacuoles in plants and yeasts, lysosomes in mammals) for degradation and recycling. Viruses are intracellular parasites that hijack their host to live. Research on regulation of the trade-off between plant cells and viruses has indicated that autophagy is an integral part of the host response to virus infection. Meanwhile, plants have evolved a diverse array of defense responses to counter pathogenic viruses. In this review, we focus on the roles of autophagy in plant virus infection and offer a glimpse of recent advances about how plant viruses evade autophagy or manipulate host autophagy pathways to complete their replication cycle.

Linking calcium and RNAi signaling in plants.

The genetic link between calcium signaling and RNA interference (RNAi) has remained undiscovered until now. A new study shows that wound-triggered calcium flux acts as an initial messenger for priming RNAi for its role in plant antiviral defense. This paves the way to investigate plant development and response to (a)biotic stresses.

Plant virus infection disrupts vacuolar acidification and autophagic degradation for the effective infection.

Vacuoles are the largest compartments in plant cells and are involved in plant development and response to abiotic and biotic stresses. Vacuolar acidification is essential for vacuoles in various physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection have never been reported. In this autophagy punctum, we discuss our recent findings about how plant viruses suppress vacuolar acidification and the degradation of autophagic bodies by directly interacting with a component of the V-ATPase to promote virus infection.

Live imaging and quantitation of insect feeding-induced Ca2+ signal using GCaMP3-based system in Nicotiana benthamiana.

Wounding evokes transient increases in cytosolic calcium (Ca2+) concentration. Visualizing real-time Ca2+ flux provides new insights into Ca2+-signaling pathways. Here, we outline a protocol to detect insect feeding-induced Ca2+ flux elevation in Nicotiana benthamiana leaves based on the GCaMP3 reporter system by Leica fluorescence stereo microscopes (LFSM). LFSM combines super-fast manual screening with high-end imaging capabilities. Through this protocol, we can clearly observe the calcium flow after aphid's piercing-sucking. Additionally, we describe a protocol to quantify Ca2+ level using LFSM. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).

A viral protein disrupts vacuolar acidification to facilitate virus infection in plants.

Vacuolar acidification is essential for vacuoles in diverse physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection remain unknown. Here, we show that Barley stripe mosaic virus (BSMV) replicase γa, but not its mutant γaR569A , directly blocks acidification of vacuolar lumen and suppresses autophagic degradation to promote viral infection in plants. These were achieved via molecular interaction between γa and V-ATPase catalytic subunit B2 (VHA-B2), leading to disruption of the interaction between VHA-B2 and V-ATPase catalytic subunit E (VHA-E), which impairs the membrane localization of VHA-B2 and suppresses V-ATPase activity. Furthermore, a mutant virus BSMVR569A with the R569A point mutation possesses less viral pathogenicity. Interestingly, multiple viral infections block vacuolar acidification. These findings reveal that functional vacuolar acidification is required for plant antiviral defense and disruption of vacuolar acidification could be a general viral counter-defense strategy employed by multiple viruses.

Transcriptional and post-transcriptional regulation of RNAi-related gene expression during plant-virus interactions.

As sessile organisms, plants encounter diverse invasions from pathogens including viruses. To survive and thrive, plants have evolved multilayered defense mechanisms to combat virus infection. RNAi, also known as RNA silencing, is an across-kingdom innate immunity and gene regulatory machinery. Molecular framework and crucial roles of RNAi in antiviral defense have been well-characterized. However, it is largely unknown that how RNAi is transcriptionally regulated to initiate, maintain and enhance cellular silencing under normal or stress conditions. Recently, insights into the transcriptional and post-transcriptional regulation of RNAi-related genes in different physiological processes have been emerging. In this review, we integrate these new findings to provide updated views on how plants modulate RNAi machinery at the (post-) transcriptional level to respond to virus infection.

Efficient and high-throughput pseudorecombinant-chimeric Cucumber mosaic virus-based VIGS in maize.

Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

A calmodulin-binding transcription factor links calcium signaling to antiviral RNAi defense in plants.

RNA interference (RNAi) is an across-kingdom gene regulatory and defense mechanism. However, little is known about how organisms sense initial cues to mobilize RNAi. Here, we show that wounding to Nicotiana benthamiana cells during virus intrusion activates RNAi-related gene expression through calcium signaling. A rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of calmodulin-binding transcription activator-3 (CAMTA3), which activates RNA-dependent RNA polymerase-6 and Bifunctional nuclease-2 (BN2) transcription. BN2 stabilizes mRNAs encoding key components of RNAi machinery, notably AGONAUTE1/2 and DICER-LIKE1, by degrading their cognate microRNAs. Consequently, multiple RNAi genes are primed for combating virus invasion. Calmodulin-, CAMTA3-, or BN2-knockdown/knockout plants show increased susceptibility to geminivirus, cucumovirus, and potyvirus. Notably, Geminivirus V2 protein can disrupt the calmodulin-CAMTA3 interaction to counteract RNAi defense. These findings link Ca2+ signaling to RNAi and reveal versatility of host antiviral defense and viral counter-defense.