Barbara Baker

Summary: The tobamovirus tomato brown rugose fruit virus (ToBRFV) has recently emerged, causing significant damage to the tomato industry in various regions worldwide, including the US. ToBRFV evades the widely used Tm‐2² resistance gene, which encodes a nucleotide‐binding leucine‐rich repeat (NLR) class immune receptor with an N‐terminal coiled‐coil (CC) domain that confers resistance to the tomato mosaic virus (ToMV). In this study, we tested a transgenic tomato line (tomato^NN) expressing the Nicotiana glutinosa N gene, which encodes an NLR with a Toll‐Interleukin 1 homology domain (TIR) at the N‐terminus, for resistance to ToBRFV. Our results demonstrate that tomato^NN is resistant to ToBRFV, evidenced by the necrotic local lesions observed on the inoculated leaves and the absence of symptoms on systemic leaves. This correlates with very low to non‐detectable virus levels in double antibody sandwich enzyme‐linked immunosorbent (DAS‐ELISA) and quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) assays. Furthermore, our findings reveal that tomato^NN is resistant to ToBRFV at 22 °C, but not at 30 °C, showing that the temperature‐sensitive nature of N‐mediated resistance also extends to ToBRFV resistance in tomato. These results highlight the significant potential of using tomato^NN to breed tomato cultivars resistant to ToBRFV, offering a new approach to managing the global pandemic caused by this emerging virus.

Plant innate immunity mediated by the nucleotide-binding leucine-rich repeat (NLR) class of immune receptors plays an important role in defense against various pathogens. Although key biochemical events involving NLR activation and signaling have been recently uncovered, we know very little about the transcriptional regulation of NLRs and their downstream signaling components. Here, we show that the Toll-Interleukin 1 receptor homology domain containing NLR (TNL) gene N (Necrosis), which confers resistance to Tobacco mosaic virus (TMV), is transcriptionally induced upon immune activation. We identified two conserved transcription factors, N required C3H Zinc finger 1 (NRZ1) and N required MYB-like transcription factor 1 (NRM1), that activate N in an immune responsive manner. Genetic analyses indicated that NRZ1 and NRM1 positively regulate coiled-coil domain-containing NLR (CNL)- and TNL-mediated immunity and function independently of the signaling component Enhanced Disease Susceptibility 1 (EDS1). Furthermore, NRZ1 functions upstream of NRM1 in cell death signaling, and their gene overexpression induces ectopic cell death and expression of NLR signaling components. Our findings uncovered a conserved transcriptional regulatory network that is central to NLR-mediated cell death and immune signaling in plants.

Understanding the efficacy of SARS-CoV-2 vaccination in people on immunosuppressive drugs, including those with rheumatoid arthritis (RA), is critical for their protection. Vaccine induced protection requires antibodies, CD4⁺ T cells, and CD8⁺ T cells, but it is unclear if these are equally affected by immunomodulatory drugs. Here, we determined how humoral and cellular SARS-CoV-2 vaccination responses differed between people with RA and controls, and which drug classes impacted these responses. Blood was collected from participants with RA on immunomodulatory drugs and controls after their second, third, and fourth SARS-CoV-2 vaccinations. Receptor binding domain (RBD)-specific antibodies were quantified by ELISA. Spike-specific memory T cells were quantitated using flow cytometry. Linear mixed models assessed the impact of age, sex, and immunomodulatory drug classes on SARS-CoV-2 vaccination responses. Compared to non-RA controls (n = 35), participants with RA on immunomodulatory drugs (n = 62) had lower anti-RBD IgG and spike-specific CD4⁺ T cell levels, but no deficits in spike-specific CD8⁺ T cells, following SARS-CoV-2 vaccination. Use of costimulation inhibitors was associated with lower humoral responses. JAK inhibitors were associated with fewer spike-specific CD4⁺ T cells. Participants with RA on immunomodulatory drugs mounted weaker responses to SARS-CoV-2 vaccination, with different drug classes impacting the cellular and humoral compartments.

Cucumber green mottle mosaic virus (CGMMV), as a typical seed-borne virus, causes costly and devastating diseases in the vegetable trade worldwide. Genetic sources for resistance to CGMMV in cucurbits are limited, and environmentally safe approaches for curbing the accumulation and spread of seed-transmitted viruses and cultivating completely resistant plants are needed. Here, we describe the design and application of RNA interference-based technologies, containing artificial microRNA (amiRNA) and synthetic trans-acting small interfering RNA (syn-tasiRNA), against conserved regions of different strains of the CGMMV genome. We used a rapid transient sensor system to identify effective anti-CGMMV amiRNAs. A virus seed transmission assay was developed, showing that the externally added polycistronic amiRNA and syn-tasiRNA can successfully block the accumulation of CGMMV in cucumber, but different virulent strains exhibited distinct influences on the expression of amiRNA due to the activity of the RNA-silencing suppressor. We also established stable transgenic cucumber plants expressing polycistronic amiRNA, which conferred disease resistance against CGMMV, and no sequence mutation was observed in CGMMV. This study demonstrates that RNA interference-based technologies can effectively prevent the occurrence and accumulation of CGMMV. The results provide a basis to establish and fine-tune approaches to prevent and treat seed-based transmission viral infections.

Main conclusion We describe a Nicotiana benthamiana system for rapid identification of artificial microRNA (amiRNA) to control cucumber green mottle mosaic virus (CGMMV) disease. Abstract Although artificial miRNA technology has been used to control other viral diseases, it has not been applied to reduce severe cucumber green mottle mosaic virus (CGMMV) disease and crop loss in the economically important cucurbits. We used our system to identify three amiRNAs targeting CGMMV RNA (amiR1-CP, amiR4-MP and amiR6-Rep) and show that their expression reduces CGMMV replication and disease in virus-infected plants. This work streamlines the process of generating amiRNA virus-resistant crops and can be broadly applied to identify active antiviral amiRNAs against a broad spectrum of viruses to control disease in diverse crops.

Summary: Plant microRNAs (miRNAs) and trans‐acting small interfering RNAs (tasiRNAs) play important roles in a variety of biological processes. Bioinformatics prediction and small RNA (sRNA) cloning are the most important approaches for identification of miRNAs and tasiRNAs and their targets. However, these approaches are not readily accessible to every researcher. Here we present SoMART, a web server for miRNA/tasiRNA analysis resources and tools, which is designed for researchers who are interested in identifying miRNAs or tasiRNAs that potentially regulate genes of interest. The server includes four sets of tools: ‘Slicer detector’ for detecting sRNAs targeting input genes, ‘dRNA mapper’ for detecting degradome (d)RNA products derived from input genes, ‘PreMIR detector’ for identifying miRNA precursors (MIRs) or tasiRNA precursor (TASs) of input sRNAs, and ‘sRNA mapper’ for mapping sRNAs onto input genes. We also developed a dRNA‐seq protocol to achieve longer dRNA reads for better characterization of miRNA precursors by dRNA mapper. To validate the server function and robustness, we installed sRNA, dRNA and collected genomic DNA or transcriptome databases from Arabidopsis and solanaceous plants, and characterized miR172‐mediated regulation of the APETALA2 gene in potato (Solanum tuberosum) and demonstrated conservation of MIR390‐triggered TAS3 in tomato (Solanum lycopersicum). More importantly, we predicted the existence of MIR482‐triggered TAS5 in tomato. We further tested and confirmed the efficiency and accuracy of the server by analyses of 21 validated miRNA targets and 115 miRNA precursors in Arabidopsis thaliana. SoMART is available at http://somart.ist.berkeley.edu.

Plant genomes contain large numbers of cell surface leucine-rich repeat (LRR) and intracellular nucleotide binding (NB)-LRR immune receptors encoded by resistance ( R ) genes that recognize specific pathogen effectors and trigger resistance responses. The unregulated expression of NB-LRR genes can trigger autoimmunity in the absence of pathogen infection and inhibit plant growth. Despite the potential serious consequence on agricultural production, the mechanisms regulating R -gene expression are not well understood. We identified microRNA (miRNA) progenitor genes precursor transcripts, and two miRNAs [nta-miR6019 (22-nt) and nta-miR6020 (21-nt)] that guide cleavage of transcripts of the Toll and Interleukin-1 receptor-NB-LRR immune receptor N from tobacco that confers resistance to tobacco mosaic virus (TMV). We further showed that cleavage by nta-miR6019 triggers RNA-dependent RNA polymerase 6- and ribonuclease Dicer-like 4-dependent biogenesis of 21-nt secondary siRNAs “in phase” with the 22-nt miR6019 cleavage site. Furthermore, we found that processing of the 22-nt nta-miR6019 depended on an asymmetric bulge caused by mismatch in the nta-miR6019 precursor. Interestingly, coexpression of N with nta-miR6019 and nta-miR6020 resulted in attenuation of N -mediated resistance to TMV, indicating that these miRNAs have functional roles in NB-LRR regulation. Using a bioinformatics approach, we identified six additional 22-nt miRNA and two 21-nt miRNA families from three Solanaceae species—tobacco, tomato, and potato. We show that members of these miRNA families cleave transcripts of predicted functional R genes and trigger production of phased secondary 21-nt siRNAs. Our results demonstrate a conserved role for miRNAs and secondary siRNAs in NB-LRR/LRR immune receptor gene regulation and pathogen resistance in Solanaceae .

MicroRNAs (miRNAs) are ubiquitous regulators of gene expression in eukaryotic organisms, which guide Argonaute proteins (AGO) to cleave target mRNA or inhibit its translation based on sequence complementarity. In plants, miRNA directed cleavage occurs on the target mRNA at about 10 to 11 nucleotide (nt) up stream to the site where the 5’ end of miRNA binds. Sequencing of the miRNA directed cleavage products (degradome) is widely employed as a way to both verify bioinformatic predictions of miRNA mediated regulation and identify novel targets of known miRNAs. Here we describe a protocol for preparation of degradome RNA complementary DNA library for high-through-put sequencing (dRNA-seq) using Illumina GA II sequencing platform, which is currently most popular and cost-effective. Using this protocol we successfully generated three dRNA-seq libraries using three solanaceae plants, including tobacco, tomato and potato. Although this protocol was developed with single-plexed adapter, it should be able to generate multiplexed libraries by replacing the 3’ adapter with multiplexing compatible 3’ adapter and replacing the PCR primer with indexed primers.

Small RNAs regulate the genome by guiding transcriptional and post-transcriptional silencing machinery to specific target sequences, including genes and transposable elements (TEs). Although miniature inverted-repeat transposable elements (MITEs) are closely associated with euchromatic genes, the broader functional impact of these short TE insertions in genes is largely unknown. We identified 22 families of MITEs in the Solanaceae (MiS1–MiS22) and found abundant MiS insertions in Solanaceae genomic DNA and expressed sequence tags (EST). Several Solanaceae MITEs generate genome changes that potentially affect gene function and regulation, most notably, a MiS insertion that provides a functionally indispensable alternative exon in the tobacco mosaic virus N resistance gene. We show that MITEs generate small RNAs that are primarily 24 nt in length, as detected by Northern blot hybridization and by sequencing small RNAs of Solanum demissum, Nicotiana glutinosa, and Nicotiana benthamiana. Additionally, we show that stable RNAi lines silencing DICER-LIKE3 (DCL3) in tobacco and RNA-dependent RNA polymerase 2 (RDR2) in potato cause a reduction in 24-nt MITE siRNAs, suggesting that, as in Arabidopsis, TE-derived siRNA biogenesis is DCL3 and RDR2 dependent. We provide evidence that DICER-LIKE4 (DCL4) may also play a role in MITE siRNA generation in the Solanaceae.