A cluster of atypical resistance genes in soybean confers broad-spectrum antiviral activity.

Soybean mosaic virus (SMV) is a severe soybean (Glycine max) pathogen. Here we characterize a soybean SMV resistance cluster (SRC) that comprises five resistance (R) genes. SRC1 encodes a Toll/interleukin-1 receptor and nucleotide-binding site (TIR-NBS [TN]) protein, SRC4 and SRC6 encode TIR proteins with a short EF-hand domain, while SRC7 and SRC8 encode TNX proteins with a noncanonical basic secretory protein (BSP) domain at their C-termini. We mainly studied SRC7, which contains a noncanonical BSP domain and gave full resistance to SMV. SRC7 possessed broad-spectrum antiviral activity toward several plant viruses including SMV, plum pox virus, potato virus Y, and tobacco mosaic virus. The TIR domain alone was both necessary and sufficient for SRC7 immune signaling, while the NBS domain enhanced its activity. Nuclear oligomerization via the interactions of both TIR and NBS domains was essential for SRC7 function. SRC7 expression was transcriptionally inducible by SMV infection and salicylic acid (SA) treatment, and SA was required for SRC7 triggered virus resistance. SRC7 expression was posttranscriptionally regulated by miR1510a and miR2109, and the SRC7-miR1510a/miR2109 regulatory network appeared to contribute to SMV-soybean interactions in both resistant and susceptible soybean cultivars. In summary, we report a soybean R gene cluster centered by SRC7 that is regulated at both transcriptional and posttranscriptional levels, possesses a yet uncharacterized BSP domain, and has broad-spectrum antiviral activities. The SRC cluster is special as it harbors several functional R genes encoding atypical TIR-NBS-LRR (TNL) type R proteins, highlighting its importance in SMV-soybean interaction and plant immunity.

Selenium-induced rhizosphere microorganisms endow salt-sensitive soybeans with salt tolerance.

Soil salinization is a prevalent abiotic stress that adversely affects soybean production. Rhizosphere microorganisms have been shown to modulate the rhizosphere microenvironment of plants, leading to improved stress resistance. Selenium is known to optimize the rhizosphere microbial community, however, it remains uncertain whether selenium-induced rhizosphere microorganisms can enhance plant salt tolerance. In this study, we selected two soybean varieties, including salt-tolerant and salt-sensitive, and conducted pot experiments to explore the impact of selenium application on the structure and composition of the rhizosphere microbial community of soybean plants under salt stress. Four salt-tolerant bacteria from salt-tolerant soybean rhizosphere soil fertilized with selenium under salt stress were isolated, and their effects on improving salt tolerance in salt-sensitive soybean were also investigated. Our results showed that selenium application enhanced soybean salt tolerance by optimizing the structure of the plant rhizosphere microbial community and improving soil enzyme activities in both salt-tolerant and salt-sensitive varieties. Moreover, compared with salt-only treatment, inoculation of the four bacteria led to a significant increase in the plant height (7.2%-19.8%), aboveground fresh weight (57.3%-73.5%), SPAD value (8.4%-30.3%), and K+ content (4.5%-12.1%) of salt-sensitive soybean, while reducing the content of proline (84.5%-94%), MDA (26.5%-49.3%), and Na+ (7.1%-21.3%). High-throughput sequencing of the 16 S ribosomal RNA gene indicated that the four bacteria played a crucial role in changing the community structure of salt-sensitive soybean and mitigating the effects of salt stress. This study highlighted the importance of selenium combined with beneficial microorganisms in the plant rhizosphere in alleviating salinity stress.

Host sunflower-induced silencing of parasitism-related genes confers resistance to invading Orobanche cumana.

Orobanche cumana is a holoparasitic plant that attaches to host-plant roots and seriously reduces the yield of sunflower (Helianthus annuus L.). Effective control methods are lacking with only a few known sources of genetic resistance. In this study, a seed-soak agroinoculation (SSA) method was established, and recombinant tobacco rattle virus vectors were constructed to express RNA interference (RNAi) inducers to cause virus-induced gene silencing (VIGS) in sunflower. A host target gene HaTubulin was systemically silenced in both leaf and root tissues by the SSA-VIGS approach. Trans-species silencing of O. cumana genes were confirmed for 10 out of 11 target genes with silencing efficiency of 23.43%-92.67%. Knockdown of target OcQR1, OcCKX5, and OcWRI1 genes reduced the haustoria number, and silencing of OcEXPA6 caused further phenotypic abnormalities such as shorter tubercles and necrosis. Overexpression of OcEXPA6 caused retarded root growth in alfalfa (Medicago sativa). The results demonstrate that these genes play an important role in the processes of O. cumana parasitism. High-throughput small RNA (sRNA) sequencing and bioinformatics analyses unveiled the distinct features of target gene-derived siRNAs in O. cumana such as siRNA transitivity, strand polarity, hotspot region, and 21/22-nt siRNA predominance, the latter of which was confirmed by Northern blot experiments. The possible RNAi mechanism is also discussed by analyzing RNAi machinery genes in O. cumana. Taken together, we established an efficient host-induced gene silencing technology for both functional genetics studies and potential control of O. cumana. The ease and effectiveness of this strategy could potentially be useful for other species provided they are amenable to SSA.

Improvement of host-induced gene silencing efficiency via polycistronic-tRNA-amiR expression for multiple target genes and characterization of RNAi mechanism in Mythimna separata.

Host-induced gene silencing (HIGS) emerged as a new strategy for pest control. However, RNAi efficiency is reported to be low in Lepidoptera, which are composed of many important crop pests. To address this, we generated transgenic plants to develop HIGS effects in a maize pest, Mythimna separata (Lepidoptera, Noctuidae), by targeting chitinase encoding genes. More importantly, we developed an artificial microRNA (amiR) based PTA (polycistronic-tRNA-amiR) system for silencing multiple target genes. Compared with hpRNA (hairpin RNA), transgenic expression of a PTA cassette including an amiR for the gut-specific dsRNA nuclease gene MsREase, resulted in improved knockdown efficiency and caused more pronounced developmental abnormalities in recipient insects. When target gene siRNAs were analysed after HIGS and direct dsRNA/siRNA feeding, common features such as sense polarity and siRNA hotspot regions were observed, however, they differed in siRNA transitivity and major 20-24nt siRNA species. Core RNAi genes were identified in M. separata, and biochemical activities of MsAGO2, MsSID1 and MsDcr2 were confirmed by EMSA (electrophoretic mobility shift assay) and dsRNA cleavage assays, respectively. Taken together, we provide compelling evidence for the existence of the RNAi mechanism in M. separata by analysis of both siRNA signatures and RNAi machinery components, and the PTA system could potentially be useful for future RNAi control of lepidopteran pests.

"Candidatus Liberibacter asiaticus" Secretes Nonclassically Secreted Proteins That Suppress Host Hypersensitive Cell Death and Induce Expression of Plant Pathogenesis-Related Proteins.

Although emerging evidence indicates that bacteria extracellularly export many cytoplasmic proteins referred to as non-classically secreted proteins (ncSecPs) for their own benefit, the mechanisms and functional significance of the ncSecPs in extracellular milieu remain elusive. "Candidatus Liberibacter asiaticus" (CLas) is a fastidious Gram-negative bacterium that causes Huanglongbing (HLB), the most globally devastating citrus disease. In this study, using the SecretomeP program coupled with an Escherichia coli alkaline phosphatase assay, we identified 27 ncSecPs from the CLas genome. Further, we demonstrated that 10 of these exhibited significantly higher levels of gene expression in citrus than in psyllid hosts, and particularly suppressed hypersensitive response (HR)-based cell death and H2O2 overaccumulation in Nicotiana benthamiana, indicating their opposing effects on early plant defenses. However, these proteins also dramatically enhanced the gene expression of pathogenesis-related 1 protein (PR-1), PR-2, and PR-5, essential components of plant defense mechanisms. Additional experiments disclosed that the increased expression of these PR genes, in particular PR-1 and PR-5, could negatively regulate HR-based cell death development and H2O2 accumulation. Remarkably, CLas infection clearly induced gene expression of PR-1, PR-2, and PR-5 in both HLB-tolerant and HLB-susceptible species of citrus plants. Taken together, we hypothesized that CLas has evolved an arsenal of ncSecPs that function cooperatively to overwhelm the early plant defenses by inducing host PR genes.IMPORTANCE In this study, we present a combined computational and experimental methodology that allows a rapid and efficient identification of the ncSecPs from bacteria, in particular the unculturable bacteria like CLas. Meanwhile, the study determined that a number of CLas ncSecPs suppressed HR-based cell death, and thus indicated a novel role for the bacterial ncSecPs in extracellular milieu. More importantly, these ncSecPs were found to suppress cell death presumably by utilizing host PR proteins. The data overall provide a novel clue to understand the CLas pathogenesis and also suggest a new way by which phytopathogens manipulate host cellular machinery to establish infection.

Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors.

BACKGROUND: RNAi (RNA interference) is a technology for silencing of target genes via sequence-specific manner. RNAi technology has been used for development of anti-pathogenic crops. In 2007, development of transgenic plants resistant to insect herbivore using RNAi technology was first reported, leading to a burst of efforts aimed at exploitation of RNAi mechanism and control strategy against variety of insect species based on this technique. Mythimna separata belongs to noctuidae family of lepidoptera and is posing threat to crops of economic importance. Recently, outbreaks of M. separata severely threatens corn production in Northern China, calling for new control approaches. RESULTS: Chitinase genes were chosen as the target genes as they were expressed predominantly in the gut tissue and were reported to be ideal silencing targets in several insect species. Interfering sequences against the target genes were cloned into the L4440 vector to produce sequence specific dsRNAs (double-stranded RNAs). Recombinant L4440 vectors were transformed into Escherichia coli strain HT115 (DE3) which was defective in dsRNA degradation activity, so preserving the dsRNA from degradation by cellular machinery. The bacteria were mixed with artificial diet and were fed to M. separata. We showed that oral delivery of bacterially expressed dsRNA would lead to RNAi effects in the recipient insect. Quantitative real-time PCR results showed that expression level of target MseChi1 and MseChi2 genes in gut tissue of M. separata were down-regulated after oral delivery of engineered bacteria expressing the corresponding dsRNA. Sequence-specific siRNA (small interfering RNA) was detected in recipient insects, supporting the existence of siRNA-mediated silencing effects in M. separata. Furthermore, knockdown of MseChi1 and MseChi2 resulted in increased mortality and reduced body weight of the feeding larvae. CONCLUSION: We reported a simple and low cost experimental procedure to silence M. separata endogenous gene expression. Our research provides both an experimental foundation for using RNAi technology to control M. separata and also a useful research tool for loss-of-function study of important developmental and regulatory genes in this insect species.

Silencing of Mythimna separata chitinase genes via oral delivery of in planta-expressed RNAi effectors from a recombinant plant virus.

OBJECTIVES: To evaluate transient expression of RNA interference (RNAi) effectors in Nicotiana benthamiana plants by using recombinant virus vectors and also oral delivery of the effectors for silencing of Mythimna separata endogenous gene expression. RESULTS: Mythimna separata is a serious pest of corn production in China. To evaluate RNAi approaches to target specific RNAs in M. separate, we cloned fragments of the M. separata chitinase sequences into a virus vector in order to produce RNAi effectors during virus infection and replication in plants. When the infected plants were fed to M. separata, expression levels of target MseChi1 and MseChi2 genes were down-regulated by 76 and 45 %, respectively, and sequence-specific siRNAs were detected in recipient insects. RNAi-based silencing of chitinase genes also led to body weight decreases by 43 %. CONCLUSION: Our research demonstrates target mRNA knockdown and suggests a promising application for controlling of M. separata by in planta expression of RNAi effectors using a recombinant plant virus.

De Novo Assembly of the Transcriptome for Oriental Armyworm Mythimna separata (Lepidoptera: Noctuidae) and Analysis on Insecticide Resistance-Related Genes.

Mythimna separata walker (Lepidoptera: Noctuidae) is a polyphagous pest of nearly 100 families of more than 300 kinds of food and industrial crops. So far, both nucleotide and protein sequence information has been rarely available in database for M. separata, strictly limiting molecular biology research in this insect species. In this study, we carried out a transcriptome sequencing for M. separata The sequencing and subsequent bioinformatics analysis yielded 69,238 unigenes, among which 45,227 unigenes were annotated to corresponding functions by blasting with high homologous genes in database, giving annotation rate of 65.32%. Several lepidopteran insects gave best matches with the transcriptome data. To gain insight into the mechanism of insecticide resistance in M. separata, 15 families of genes encoding insecticide resistance-related proteins were investigated. Substantial numbers of unigenes in these families were identified in the transcriptome data, and 17 out of 21 selected unigenes were successfully amplified. Expressions of most of these genes were detected at larval stages and in gut tissue, as was consistent with their putative involvement in insecticide resistance. Our study provides most comprehensive transcriptome data for M. separata to date, and also provides reference sequence information for other Noctuidae family insects.

An S-domain receptor-like kinase, OsSIK2, confers abiotic stress tolerance and delays dark-induced leaf senescence in rice.

Receptor-like kinases play important roles in plant development and defense responses; however, their functions in other processes remain unclear. Here, we report that OsSIK2, an S-domain receptor-like kinase from rice (Oryza sativa), is involved in abiotic stress and the senescence process. OsSIK2 is a plasma membrane-localized protein with kinase activity in the presence of Mn(2+). OsSIK2 is expressed mainly in rice leaf and sheath and can be induced by NaCl, drought, cold, dark, and abscisic acid treatment. Transgenic plants overexpressing OsSIK2 and mutant sik2 exhibit enhanced and reduced tolerance to salt and drought stress, respectively, compared with the controls. Interestingly, a truncated version of OsSIK2 without most of the extracellular region confers higher salt tolerance than the full-length OsSIK2, likely through the activation of different sets of downstream genes. Moreover, seedlings of OsSIK2-overexpressing transgenic plants exhibit early leaf development and a delayed dark-induced senescence phenotype, while mutant sik2 shows the opposite phenotype. The downstream PR-related genes specifically up-regulated by full-length OsSIK2 or the DREB-like genes solely enhanced by truncated OsSIK2 are all induced by salt, drought, and dark treatments. These results indicate that OsSIK2 may integrate stress signals into a developmental program for better adaptive growth under unfavorable conditions. Manipulation of OsSIK2 should facilitate the improvement of production in rice and other crops.

Transcriptome Analysis for Salt-Responsive Genes in Two Different Alfalfa (Medicago sativa L.) Cultivars and Functional Analysis of MsHPCA1.

Alfalfa (Medicago sativa L.) is an important forage legume and soil salinization seriously affects its growth and yield. In a previous study, we identified a salt-tolerant variety 'Gongnong NO.1' and a salt-sensitive variety 'Sibeide'. To unravel the molecular mechanism involved in salt stress, we conducted transcriptomic analysis on these two cultivars grown under 0 and 250 mM NaCl treatments for 0, 12, and 24 h. Totals of 336, and 548 differentially expressed genes (DEGs) in response to NaCl were, respectively, identified in the 'Gongnong NO.1' and 'Sibeide' varieties. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analysis showed that the DEGs were classified in carbohydrate metabolism, energy production, transcription factor, and stress-associated pathway. Expression of MsHPCA1, encoding a putative H2O2 receptor, was responsive to both NaCl and H2O2 treatment. MsHPCA1 was localized in cell membrane and overexpression of MsHPCA1 in alfalfa increased salt tolerance and H2O2 content. This study will provide new gene resources for the improvement in salt tolerance in alfalfa and legume crops, which has important theoretical significance and potential application value.

Development of an NLR-ID Toolkit and Identification of Novel Disease-Resistance Genes in Soybean.

The recognition of pathogen effectors through the nucleotide-binding leucine-rich repeat receptor (NLR) family is an important component of plant immunity. In addition to typical domains such as TIR, CC, NBS, and LRR, NLR proteins also contain some atypical integrated domains (IDs), the roles of which are rarely investigated. Here, we carefully screened the soybean (Glycine max) genome and identified the IDs that appeared in the soybean TNL-like proteins. Our results show that multiple IDs (36) are widely present in soybean TNL-like proteins. A total of 27 Gm-TNL-ID genes (soybean TNL-like gene encoding ID) were cloned and their antiviral activity towards the soybean mosaic virus (SMV)/tobacco mosaic virus (TMV) was verified. Two resistance (R) genes, SRA2 (SMV resistance gene contains AAA_22 domain) and SRZ4 (SMV resistance gene contains zf-RVT domain), were identified to possess broad-spectrum resistance characteristics towards six viruses including SMV, TMV, plum pox virus (PPV), cabbage leaf curl virus (CaLCuV), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV). The effects of Gm-TNL-IDX (the domain of the Gm-TNL-ID gene after the TN domain) on the antiviral activity of a R protein SRC7TN (we previously reported the TN domain of the soybean broad-spectrum resistance gene SRC7) were validated, and most of Gm-TNL-IDX inhibits antiviral activity mediated by SRC7TN, possibly through intramolecular interactions. Yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that seven Gm-TNL-IDX interacted with SMV-component proteins. Truncation analysis on a broad-spectrum antiviral protein SRZ4 indicated that SRZ4TIR is sufficient to mediate antiviral activity against SMV. Soybean cDNA library screening on SRZ4 identified 48 interacting proteins. In summary, our results indicate that the integration of IDs in soybean is widespread and frequent. The NLR-ID toolkit we provide is expected to be valuable for elucidating the functions of atypical NLR proteins in the plant immune system and lay the foundation for the development of engineering NLR for plant-disease control in the future.

Large-scale mRNA transfer between Haloxylon ammodendron (Chenopodiaceae) and herbaceous root holoparasite Cistanche deserticola (Orobanchaceae).

Exchanges of mRNA were shown between host and stem parasites but not root parasites. Cistanche deserticola (Orobanchaceae) is a holoparasitic herb which parasitizes on the roots of woody plant Haloxylon ammodendron (Chenopodiaceae). We used transcriptome sequencing and bioinformatic analyses to identify nearly ten thousand mobile mRNAs. Transcript abundance appears to be a driving force for transfer event and mRNA exchanges occur through haustorial junction. Mobility of selected mRNAs was confirmed in situ and in sunflower-Orobanche cumana heterologous parasitic system. Four C. deserticola →H. ammodendron mobile mRNAs appear to facilitate haustorium development. Of interest, two mobile mRNAs of putative resistance genes CdNLR1 and CdNLR2 cause root-specific hypersensitive response and retard parasite development, which might contribute to parasitic equilibrium. The present study provides evidence for the large-scale mRNA transfer event between a woody host and a root parasite, and demonstrates the functional relevance of six C. deserticola genes in host-parasite interactions.

Epigenetic regulation of phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 gene expression in prostate cancer cells.

Aberrant up-regulation of P-Rex1 expression plays important roles in cancer progression and metastasis. The present study investigated the regulatory mechanism underlying P-Rex1 gene expression in prostate cancer cells. We showed that P-Rex1 expression was much higher in metastatic prostate cancer cells than in prostate epithelial cells and non-metastatic prostate cancer cells. Histone deacetylase (HDAC) inhibitors or silence of endogenous HDAC1 and HDAC2 markedly elevated P-Rex1 transcription in non-metastatic prostate cancer cells, whereas overexpression of recombinant HDAC1 in metastatic prostate cancer cells suppressed P-Rex1 expression. HDAC inhibitor trichostatin A (TSA) also significantly increased P-Rex1 promoter activity and caused acetylated histones to accumulate and associate with the P-Rex1 promoter. One Sp1 site, essential for basal promoter activity, was identified as critical for the TSA effect. TSA treatment did not alter the DNA-binding activity of Sp1 toward the P-Rex1 promoter; however, it facilitated the dissociation of the repressive HDAC1 and HDAC2 from the Sp1 binding region. Interestingly, HDAC1 association with Sp1 and with the P-Rex1 promoter were much weaker in metastatic prostate cancer PC-3 cells than in non-metastatic prostate cancer cells, and HDAC inhibitors only had very modest stimulatory effects on P-Rex1 promoter activity and P-Rex1 expression in PC-3 cells. Altogether, our studies demonstrate that HDACs could regulate P-Rex1 gene transcription by interaction with Sp1 and by region-specific changes in histone acetylation within the P-Rex1 promoter. Disassociation of HDACs from Sp1 on the P-Rex1 promoter may contribute to aberrant up-regulation of P-Rex1 in cancer.

RNA 5-Methylcytosine Modification Regulates Vegetative Development Associated with H3K27 Trimethylation in Arabidopsis.

Methylating RNA post-transcriptionally is emerging as a significant mechanism of gene regulation in eukaryotes. The crosstalk between RNA methylation and histone modification is critical for chromatin state and gene expression in mammals. However, it is not well understood mechanistically in plants. Here, the authors report a genome-wide correlation between RNA 5-cytosine methylation (m5 C) and histone 3 lysine27 trimethylation (H3K27me3) in Arabidopsis. The plant-specific Polycomb group (PcG) protein EMBRYONIC FLOWER1 (EMF1) plays dual roles as activators or repressors. Transcriptome-wide RNA m5 C profiling revealed that m5 C peaks are mostly enriched in chromatin regions that lacked H3K27me3 in both wild type and emf1 mutants. EMF1 repressed the expression of m5 C methyltransferase tRNA specific methyltransferase 4B (TRM4B) through H3K4me3, independent of PcG-mediated H3K27me3 mechanism. The 5-Cytosine methylation on targets is increased in emf1 mutants, thereby decreased the mRNA transcripts of photosynthesis and chloroplast genes. In addition, impairing EMF1 activity reduced H3K27me3 levels of PcG targets, such as starch genes, which are de-repressed in emf1 mutants. Both EMF1-mediated promotion and repression of gene activities via m5 C and H3K27me3 are required for normal vegetative growth. Collectively, t study reveals a previously undescribed epigenetic mechanism of RNA m5 C modifications and histone modifications to regulate gene expression in eukaryotes.

Decoy engineering of the receptor-like cytoplasmic kinase StPBS1 to defend against virus infection in potato.

Potato virus Y (PVY) is an important pathogen of potato (Solanum tuberosum). Although the PBS1-RPS5 immune system is well documented in Arabidopsis thaliana, it has not been reported in potato. In Arabidopsis, the bacterial effector AvrPphB cleaves AtPBS1 to trigger an immune response. Here, we show that the AvrPphB-triggered immune response is mediated by StPBS1, a close homologue of AtPBS1 in potato. However, downstream signalling of StPBS1 was mediated by unknown resistance (R) proteins other than potato orthologues of AtRPS5 and HvPBR1, which is important for HvPBS1 signalling in barley. Immune signalling of StPBS1 is mediated by the AvrPphB C-terminal cleavage domain and an STKPQ motif, in contrast to AtPBS1-mediated immunity in which both AvrPphB cleavage fragments and an SEMPH motif are essential. The cleavage sequence of AvrPphB in StPBS1 was replaced with that of the PVY NIa-Pro protease to obtain StPBS1NIa . StPBS1NIa overexpression potato displayed stronger immunity to PVY infection than did the StPBS1 transgenic lines. StPBS1NIa was cleaved at the expected target site by NIa-Pro protease from PVY. Thus, we characterized the function of StPBS1 in potato immunity and provide a biotechnology control method for PVY via transformation of decoy-engineered StPBS1NIa .

Expression of an Antiviral Gene GmRUN1 from Soybean Is Regulated via Intron-Mediated Enhancement (IME).

Most of R (resistance) genes encode the protein containing NBS-LRR (nucleotide binding site and leucine-rich repeat) domains. Here, N. benthamiana plants were used for transient expression assays at 3-4 weeks of age. We identified a TNL (TIR-NBS-LRR) encoding gene GmRUN1 that was resistant to both soybean mosaic virus (SMV) and tobacco mosaic virus (TMV). Truncation analysis indicated the importance of all three canonical domains for GmRUN1-mediated antiviral activity. Promoter-GUS analysis showed that GmRUN1 expression is inducible by both salicylic acid (SA) and a transcription factor GmDREB3 via the cis-elements as-1 and ERE (ethylene response element), which are present in its promoter region. Interestingly, GmRUN1 gDNA (genomic DNA) shows higher viral resistance than its cDNA (complementary DNA), indicating the existence of intron-mediated enhancement (IME) for GmRUN1 regulation. We provided evidence that intron2 of GmRUN1 increased the mRNA level of native gene GmRUN1, a soybean antiviral gene SRC7 and also a reporter gene Luciferase, indicating the general transcriptional enhancement of intron2 in different genes. In summary, we identified an antiviral TNL type soybean gene GmRUN1, expression of which was regulated at different layers. The investigation of GmRUN1 gene regulatory network would help to explore the mechanism underlying soybean-SMV interactions.

Tomato SlBL4 plays an important role in fruit pedicel organogenesis and abscission.

Abscission, a cell separation process, is an important trait that influences grain and fruit yield. We previously reported that BEL1-LIKE HOMEODOMAIN 4 (SlBL4) is involved in chloroplast development and cell wall metabolism in tomato fruit. In the present study, we showed that silencing SlBL4 resulted in the enlargement and pre-abscission of the tomato (Solanum lycopersicum cv. Micro-TOM) fruit pedicel. The anatomic analysis showed the presence of more epidermal cell layers and no obvious abscission zone (AZ) in the SlBL4 RNAi lines compared with the wild-type plants. RNA-seq analysis indicated that the regulation of abscission by SlBL4 was associated with the altered abundance of genes related to key meristems, auxin transporters, signaling components, and cell wall metabolism. Furthermore, SlBL4 positively affected the auxin concentration in the abscission zone. A dual-luciferase reporter assay revealed that SlBL4 activated the transcription of the JOINTLESS, OVATE, PIN1, and LAX3 genes. We reported a novel function of SlBL4, which plays key roles in fruit pedicel organogenesis and abscission in tomatoes.

Melatonin Enhances Seed Germination and Seedling Growth of Medicago sativa Under Salinity via a Putative Melatonin Receptor MsPMTR1.

Alfalfa (Medicago sativa L.) is an important forage crop, and salt stress is a major limiting factor in its yield. Melatonin (MT) is a multi-regulatory molecule in plants. We showed that basal MT content was positively correlated with the salt tolerance degree of different alfalfa varieties. MT and its precursor 5-HT fully recovered seed germination while partially ameliorated seedling growth of salt-stressed alfalfa. The 5-HT showed some divergent effects from MT with regards to growth amelioration under salinity. Salt stress caused stunted plant growth in soil culture, while MT ameliorated it by elevating plant height, fresh weight, branching number, and chlorophyll content. Silencing of a putative MT receptor, MsPMTR1, which was shown to be membrane-localized, abolished the ameliorative effects of MT on salt-stressed alfalfa seedling growth, while overexpression of MsPMTR1 improved plant growth under salt stress. The RNA sequencing analysis showed that nine pathway genes were specifically induced by MT treatment compared with salt stress. These MT-responsive differentially expressed genes include basal metabolic pathway genes, such as "ribosome, elongation factor," "sugar and lipid metabolism," and "photosynthesis" and stress-related genes encoding "membrane integrity" related proteins, heat shock protein, peroxidase/oxidoreductase, and protease. Several abiotic stress response-related genes, such as DRE, ARF, HD-ZF, MYB, and REM were repressed by NaCl treatment while induced by MT treatment. In summary, we demonstrated the importance of MsPMTR1 in MT-mediated salt tolerance in alfalfa, and we also analyzed the regulatory mechanism of MT during alfalfa seed germination under salt stress.

Synthesis of Full-Length cDNA Infectious Clones of Soybean Mosaic Virus and Functional Identification of a Key Amino Acid in the Silencing Suppressor Hc-Pro.

Soybean mosaic virus (SMV), which belongs to the Potyviridae, causes significant reductions in soybean yield and seed quality. In this study, both tag-free and reporter gene green fluorescent protein (GFP)-containing infectious clones for the SMV N1 strain were constructed by Gibson assembly and with the yeast homologous recombination system, respectively. Both infectious clones are suitable for agroinfiltration on the model host N. benthamiana and show strong infectivity for the natural host soybean and several other legume species. Both infectious clones were seed transmitted and caused typical virus symptoms on seeds and progeny plants. We used the SMV-GFP infectious clone to further investigate the role of key amino acids in the silencing suppressor helper component-proteinase (Hc-Pro). Among twelve amino acid substitution mutants, the co-expression of mutant 2-with an Asparagine→Leucine substitution at position 182 of the FRNK (Phe-Arg-Asn-Lys) motif-attenuated viral symptoms and alleviated the host growth retardation caused by SMV. Moreover, the Hc-Prom2 mutant showed stronger oligomerization than wild-type Hc-Pro. Taken together, the SMV infectious clones will be useful for studies of host-SMV interactions and functional gene characterization in soybeans and related legume species, especially in terms of seed transmission properties. Furthermore, the SMV-GFP infectious clone will also facilitate functional studies of both virus and host genes in an N. benthamiana transient expression system.

Ethylene Enhances Seed Germination and Seedling Growth Under Salinity by Reducing Oxidative Stress and Promoting Chlorophyll Content via ETR2 Pathway.

Alfalfa (Medicago sativa L.) is an important forage, and salinity is a major stress factor on its yield. In this study, we show that osmotic stress retards alfalfa seedling growth, while ionic/oxidative stress reduces its seed germination. Ethylene treatment can recover the germination rate of alfalfa seeds under salt stress, while ethylene inhibitor silver thiosulfate exacerbates salt effects. ETH reduces the accumulation of MDA and H2O2 and increases POD activity. ETH and ACC improve the salt tolerance of alfalfa by increasing proline content under salt stress. In contrast, STS inhibits alfalfa seed germination by reducing POD activity. NaCl treatment reduces chlorophyll content in alfalfa leaves, while ETH and ACC can increase the chlorophyll content and promote seedling growth. ETH promotes the growth of alfalfa in saline condition by reducing the expression of MsACO and MsERF8 genes, while increases its germination rate by upregulating MsERF11 gene. Silencing of MsETR2, a putative ethylene receptor gene in alfalfa, abolishes ethylene triggered tolerance to salt stress. In summary, we show that ethylene improves salt tolerance in alfalfa via MsETR2 dependent manner, and we also analyze the regulatory mechanism of ethylene during germination of alfalfa seeds under salt stress.