Proxitome profiling reveals a conserved SGT1-NSL1 signaling module that activates NLR-mediated immunity.

SGT1 is a highly conserved eukaryotic protein that plays a vital role in the growth, development, and immunity in both animals and plants. Although some SGT1 interactors have been identified, the molecular regulatory network of SGT1 remains unclear. SGT1 serves as a co-chaperone to stabilize protein complexes such as the nucleotide-binding leucine-rich repeat (NLR) class of immune receptors, thereby positively regulating plant immunity. SGT1 has also been found to be associated with the SKP1-Cullin-F-box (SCF) E3 ubiquitin ligase complex. However, whether SGT1 targets immune repressors to coordinate plant immune activation remains elusive. Here, we constructed a toolbox for TurboID- and split-TurboID-based proximity labeling (PL) assays in Nicotiana benthamiana. We used the PL toolbox to explore the SGT1 interactome during pre- and post-immune activation. The comprehensive SGT1 interactome network that we identified highlights a dynamic shift from proteins associated with plant development to those linked with plant immune responses. SGT1 interacts with Necrotic Spotted Lesion 1 (NSL1) that negatively regulates salicylic acid (SA)-mediated defense by interfering with the nucleocytoplasmic trafficking of Non-expressor of Pathogenesis-Related Genes 1 (NPR1) during N NLR-mediated response to tobacco mosaic virus (TMV). SGT1 promotes the SCF-dependent degradation of NSL1 to facilitate immune activation, while salicylate-induced protein kinase (SIPK)-mediated phosphorylation of SGT1 further potentiates this process. Besides N NLR, NSL1 also functions in several other NLR-mediated immunity. Our study unveils the regulatory landscape of SGT1 and reveals a novel SGT1-NSL1 signaling module that orchestrates plant innate immunity.

A potyvirus provides an efficient viral vector for gene expression and functional studies in Asteraceae plants.

Plant virus-derived vectors are rapid and cost-effective for protein expression and gene functional studies in plants, particularly for species that are difficult to genetically transform. However, few efficient viral vectors are available for functional studies in Asteraceae plants. Here, we identified a potyvirus named zinnia mild mottle virus (ZiMMV) from common zinnia (Zinnia elegans Jacq.) through next-generation sequencing. Using a yeast homologous recombination strategy, we established a full-length infectious cDNA clone of ZiMMV under the control of the cauliflower mosaic virus 35S promoter. Furthermore, we developed an efficient expression vector based on ZiMMV for the persistent and abundant expression of foreign proteins in the leaf, stem, root, and flower tissues with mild symptoms during viral infection in common zinnia. We showed that the ZiMMV-based vector can express ZeMYB9, which encodes a transcript factor inducing dark red speckles in leaves and flowers. Additionally, the expression of a gibberellic acid (GA) biosynthesis gene from the ZiMMV vector substantially accelerated plant height growth, offering a rapid and cost-effective method. In summary, our work provides a powerful tool for gene expression, functional studies, and genetic improvement of horticultural traits in Asteraceae plant hosts.

The MAPK-Alfin-like 7 module negatively regulates ROS scavenging genes to promote NLR-mediated immunity.

Nucleotide-binding leucine-rich repeat (NLR) receptor-mediated immunity includes rapid production of reactive oxygen species (ROS) and transcriptional reprogramming, which is controlled by transcription factors (TFs). Although some TFs have been reported to participate in NLR-mediated immune response, most TFs are transcriptional activators, and whether and how transcriptional repressors regulate NLR-mediated plant defenses remains largely unknown. Here, we show that the Alfin-like 7 (AL7) interacts with N NLR and functions as a transcriptional repressor. Knockdown and knockout of AL7 compromise N NLR-mediated resistance against tobacco mosaic virus, whereas AL7 overexpression enhances defense, indicating a positive regulatory role for AL7 in immunity. AL7 binds to the promoters of ROS scavenging genes to inhibit their transcription during immune responses. Mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK) directly interact with and phosphorylate AL7, which impairs the AL7-N interaction and enhances its DNA binding activity, which promotes ROS accumulation and enables immune activation. In addition to N, AL7 is also required for the function of other Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeats (TNLs) including Roq1 and RRS1-R/RPS4. Our findings reveal a hitherto unknown MAPK-AL7 module that negatively regulates ROS scavenging genes to promote NLR-mediated immunity.

Coat proteins of necroviruses target 14-3-3a to subvert MAPKKKα-mediated antiviral immunity in plants.

Mitogen-activated protein kinase (MAPK) cascades play an important role in innate immunity against various pathogens in plants and animals. However, we know very little about the importance of MAPK cascades in plant defense against viral pathogens. Here, we used a positive-strand RNA necrovirus, beet black scorch virus (BBSV), as a model to investigate the relationship between MAPK signaling and virus infection. Our findings showed that BBSV infection activates MAPK signaling, whereas viral coat protein (CP) counteracts MAPKKKα-mediated antiviral defense. CP does not directly target MAPKKKα, instead it competitively interferes with the binding of 14-3-3a to MAPKKKα in a dose-dependent manner. This results in the instability of MAPKKKα and subversion of MAPKKKα-mediated antiviral defense. Considering the conservation of 14-3-3-binding sites in the CPs of diverse plant viruses, we provide evidence that 14-3-3-MAPKKKα defense signaling module is a target of viral effectors in the ongoing arms race of defense and viral counter-defense.

SAMDC3 enhances resistance to Barley stripe mosaic virus by promoting the ubiquitination and proteasomal degradation of viral γb protein.

Posttranslational modifications (PTMs) play important roles in virus-host interplay. We previously demonstrated that Barley stripe mosaic virus (BSMV) γb protein is phosphorylated by different host kinases to support or impede viral infection. However, whether and how other types of PTMs participate in BSMV infection remains to be explored. Here, we report that S-adenosylmethionine decarboxylase 3 (SAMDC3) from Nicotiana benthamiana or wheat (Triticum aestivum) interacts with γb. BSMV infection induced SAMDC3 expression. Overexpression of SAMDC3 led to the destabilization of γb and reduction in viral infectivity, whereas knocking out NbSAMDC3 increased susceptibility to BSMV. NbSAMDC3 positively regulated the 26S proteasome-mediated degradation of γb via its PEST domain. Further mechanistic studies revealed that γb can be ubiquitinated in planta and that NbSAMDC3 promotes the proteasomal degradation of γb by increasing γb ubiquitination. We also found evidence that ubiquitination occurs at nonlysine residues (Ser-133 and Cys-144) within γb. Together, our results provide a function for SAMDC3 in defence against BSMV infection through targeting of γb abundance, which contributes to our understanding of how a plant host deploys the ubiquitin-proteasome system to mount defences against viral infections.

Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture.

Genome editing provides novel strategies for improving plant traits but mostly relies on conventional plant genetic transformation and regeneration procedures, which can be inefficient. In this study, we have engineered a Barley stripe mosaic virus-based sgRNA delivery vector (BSMV-sg) that is effective in performing heritable genome editing in Cas9-transgenic wheat plants. Mutated progenies were present in the next generation at frequencies ranging from 12.9% to 100% in three different wheat varieties, and 53.8%-100% of mutants were virus free. We also achieved multiplex mutagenesis in progeny using a pool of BSMV-sg vectors harboring different sgRNAs. Furthermore, we devised a virus-induced transgene-free editing procedure to generate Cas9-free wheat mutants by crossing BSMV-infected Cas9-transgenic wheat pollen with wild-type wheat. Our study provides a robust, convenient, and tissue culture-free approach for genome editing in wheat through virus infection.

Proximity labeling: an emerging tool for probing in planta molecular interactions.

Protein-protein interaction (PPI) networks are key to nearly all aspects of cellular activity. Therefore, the identification of PPIs is important for understanding a specific biological process in an organism. Compared with conventional methods for probing PPIs, the recently described proximity labeling (PL) approach combined with mass spectrometry (MS)-based quantitative proteomics has emerged as a powerful approach for characterizing PPIs. However, the application of PL in planta remains in its infancy. Here, we summarize recent progress in PL and its potential utilization in plant biology. We specifically summarize advances in PL, including the development and comparison of different PL enzymes and the application of PL for deciphering various molecular interactions in different organisms with an emphasis on plant systems.