A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Mediator kinase subunit CDK8 phosphorylates transcription factor TCP15 during tomato pollen development.

Pollen development in flowering plants has strong implications for reproductive success. Pollen DNA can be targeted to improve plant traits for yield and stress tolerance. In this study, we demonstrated that the Mediator subunit CYCLIN-DEPENDENT KINASE 8 (CDK8) is a key modulator of pollen development in tomato (Solanum lycopersicum). SlCDK8 knockout led to significant decreases in pollen viability, fruit yield, and fruit seed number. We also found that SlCDK8 directly interacts with transcription factor TEOSINTE BRANCHED1-CYCLOIDEA-PCF15 (SlTCP15) using yeast two-hybrid screens. We subsequently showed that SlCDK8 phosphorylates Ser 187 of SlTCP15 to promote SlTCP15 stability. Phosphorylated TCP15 directly bound to the TGGGCY sequence in the promoters of DYSFUNCTIONAL TAPETUM 1 (SlDYT1) and MYB DOMAIN PROTEIN 103 (SlMYB103), which are responsible for pollen development. Consistently, disruption of SlTCP15 resembled slcdk8 tomato mutants. In sum, our work identified a new substrate of Mediator CDK8 and revealed an important regulatory role of SlCDK8 in pollen development via cooperation with SlTCP15.

ZmC2H2-149 negatively regulates drought tolerance by repressing ZmHSD1 in maize.

Drought is a major abiotic stress that limits maize production worldwide. Therefore, it is of great importance to improve drought tolerance in crop plants for sustainable agriculture. In this study, we examined the roles of Cys2 /His2 zinc-finger-proteins (C2H2-ZFPs) in maize's drought tolerance as C2H2-ZFPs have been implicated for plant stress tolerance. By subjecting 150 Ac/Ds mutant lines to drought stress, we successfully identified a Ds-insertion mutant, zmc2h2-149, which shows increased tolerance to drought stress. Overexpression of ZmC2H2-149 in maize led to a decrease in both drought tolerance and crop yield. DAP-Seq, RNA-Seq, Y1H and LUC assays additionally showed that ZmC2H2-149 directly suppresses the expression of a positive drought tolerance regulator, ZmHSD1 (hydroxysteroid dehydrogenase 1). Consistently, the zmhsd1 mutants exhibited decreased drought tolerance and grain yield under water deficit conditions compared to their respective wild-type plants. Our findings thus demonstrated that ZmC2H2-149 can regulate ZmHSD1 for drought stress tolerance in maize, offering valuable theoretical and genetic resources for maize breeding programmes that aim for improving drought tolerance.

ZmELF6-ZmPRR37 module regulates maize flowering and salt response.

The control of flowering time in maize is crucial for reproductive success and yield, and it can be influenced by environmental stresses. Using the approaches of Ac/Ds transposon and transposable element amplicon sequencing techniques, we identified a Ds insertion mutant in the ZmPRR37 gene. The Ds insertion showed a significant correlation with days to anthesis. Further research indicated that ZmPRR37-CR knockout mutants exhibited early flowering, whereas ZmPRR37-overexpression lines displayed delayed flowering compared to WT under long-day (LD) conditions. We demonstrated that ZmPRR37 repressed the expression of ZmNF-YC2 and ZmNF-YA3 to delay flowering. Association analysis revealed a significant correlation between flowering time and a SNP2071-C/T located upstream of ZmPRR37. The SNP2071-C/T impacted the binding capacity of ZmELF6 to the promoter of ZmPRR37. ZmELF6 also acted as a flowering suppressor in maize under LD conditions. Notably, our study unveiled that ZmPRR37 can enhance salt stress tolerance in maize by directly regulating the expression of ABA-responsive gene ZmDhn1. ZmDhn1 negatively regulated maize salt stress resistance. In summary, our findings proposed a novel pathway for regulating photoperiodic flowering and responding to salt stress based on ZmPRR37 in maize, providing novel insights into the integration of abiotic stress signals into floral pathways.

Molecular mechanism analysis of ZmRL6 positively regulating drought stress tolerance in maize.

MYB-related genes, a subclass of MYB transcription factor family, have been documented to play important roles in biological processes such as secondary metabolism and stress responses that affect plant growth and development. However, the regulatory roles of MYB-related genes in drought stress response remain unclear in maize. In this study, we discovered that a 1R-MYB gene, ZmRL6, encodes a 96-amino acid protein and is highly drought-inducible. We also found that it is conserved in both barley (Hordeum vulgare L.) and Aegilops tauschii. Furthermore, we observed that overexpression of ZmRL6 can enhance drought tolerance while knock-out of ZmRL6 by CRISPR-Cas9 results in drought hypersensitivity. DAP-seq analyses additionally revealed the ZmRL6 target genes mainly contain ACCGTT, TTACCAAAC and AGCCCGAG motifs in their promoters. By combining RNA-seq and DAP-seq results together, we subsequently identified eight novel target genes of ZmRL6 that are involved in maize's hormone signal transduction, sugar metabolism, lignin synthesis, and redox signaling/oxidative stress. Collectively, our data provided insights into the roles of ZmRL6 in maize's drought response.

BIN2 phosphorylates the Thr280 of CO to restrict its function in promoting Arabidopsis flowering.

CONSTANS (CO) is a central regulator of floral initiation in response to photoperiod. In this study, we show that the GSK3 kinase BIN2 physically interacts with CO and the gain-of-function mutant bin2-1 displays late flowering phenotype through down-regulation of FT transcription. Genetic analyses show that BIN2 genetically acts upstream of CO in regulating flowering time. Further, we illustrate that BIN2 phosphorylates the Thr280 residue of CO. Importantly, the BIN2 phosphorylation of Thr280 residue restricts the function of CO in promoting flowering through affecting its DNA-binding activity. Moreover, we reveal that the N-terminal part of CO harboring the B-Box domain mediates the interaction of both CO-CO and BIN2-CO. We find that BIN2 inhibits the formation of CO dimer/oligomer. Taken together, this study reveals that BIN2 regulates flowering time through phosphorylating the Thr280 of CO and inhibiting the CO-CO interaction in Arabidopsis.

The Jasmine (Jasminum sambac) Genome Provides Insight into the Biosynthesis of Flower Fragrances and Jasmonates.

Jasminum sambac (jasmine flower), a world-renowned plant appreciated for its exceptional flower fragrance, is of cultural and economic importance. However, the genetic basis of its fragrance is largely unknown. Here, we present the first de novogenome assembly of J. sambac with 550.12 Mb (scaffold N50 = 40.10 Mb) assembled into 13 pseudochromosomes. Terpene synthase (TPS) genes associated with flower fragrance are considerably amplified in the form of gene clusters through tandem duplications in the genome. Gene clusters within the salicylic acid/benzoic acid/theobromine (SABATH) and benzylalcohol O-acetyltransferase/anthocyanin O-hydroxycinnamoyltransferases/anthranilate N-hydroxycinnamoyl/benzoyltransferase/deacetylvindoline 4-O-acetyltransferase (BAHD) superfamilies were identified to be related to the biosynthesis of phenylpropanoid/benzenoid compounds. Several key genes involved in jasmonate biosynthesis were duplicated, causing an increase in copy numbers. In addition, multi-omics analyses identified various aromatic compounds and many genes involved in fragrance biosynthesis pathways. Furthermore, the roles of JsTPS3 in ß-ocimene biosynthesis, as well as JsAOC1 and JsAOS in jasmonic acid biosynthesis, were functionally validated. The genome assembled in this study for J. sambac offers a basic genetic resource for studying floral scent and jasmonate biosynthesis, and provides a foundation for functional genomic research and variety improvements in Jasminum.

A natural promoter variation of SlBBX31 confers enhanced cold tolerance during tomato domestication.

Cold stress affects crop growth and productivity worldwide. Understanding the genetic basis of cold tolerance in germplasms is critical for crop improvement. Plants can coordinate environmental stimuli of light and temperature to regulate cold tolerance. However, it remains unknown which gene in germplasms could have such function. Here, we utilized genome-wide association study (GWAS) to investigate the cold tolerance of wild and cultivated tomato accessions and discovered that increased cold tolerance is accompanied with tomato domestication. We further identified a 27-bp InDel in the promoter of the CONSTANS-like transcription factor (TF) SlBBX31 is significantly linked with cold tolerance. Coincidentally, a key regulator of light signalling, SlHY5, can directly bind to the SlBBX31 promoter to activate SlBBX31 transcription while the 27-bp InDel can prevent S1HY5 from transactivating SlBBX31. Parallel to these findings, we observed that the loss of function of SlBBX31 results in impaired tomato cold tolerance. SlBBX31 can also modulate the cold-induced expression of several ERF TFs including CBF2 and DREBs. Therefore, our study has uncovered that SlBBX31 is possibly selected during tomato domestication for cold tolerance regulation, providing valuable insights for the development of hardy tomato varieties.

Advances in mass spectrometry-based phosphoproteomics for elucidating abscisic acid signaling and plant responses to abiotic stress.

Abiotic stresses have significant impacts on crop yield and quality. Even though significant efforts during the past decade have been devoted to uncovering the core signaling pathways associated with the phytohormone abscisic acid (ABA) and abiotic stress in plants, abiotic stress signaling mechanisms in most crops remain largely unclear. The core components of the ABA signaling pathway, including early events in the osmotic stress-induced phosphorylation network, have recently been elucidated in Arabidopsis with the aid of phosphoproteomics technologies. We now know that SNF1-related kinases 2 (SnRK2s) are not only inhibited by the clade A type 2C protein phosphatases (PP2Cs) through dephosphorylation, but also phosphorylated and activated by upstream mitogen-activated protein kinase kinase kinases (MAP3Ks). Through describing the course of studies to elucidate abiotic stress and ABA signaling, we will discuss how we can take advantage of the latest innovations in mass-spectrometry-based phosphoproteomics and structural proteomics to boost our investigation of plant regulation and responses to ABA and abiotic stress.

Genome-wide association study of eigenvectors provides genetic insights into selective breeding for tomato metabolites.

BACKGROUND: Long-term domestication and intensive breeding of crop plants aim to establish traits desirable for human needs, and characteristics related to yield, disease resistance, and postharvest storage have traditionally received considerable attention. These processes have led also to negative consequences, as is the case of loss of variants controlling fruit quality, for instance in tomato. Tomato fruit quality is directly associated to metabolite content profiles; however, a full understanding of the genetics affecting metabolite content during tomato domestication and improvement has not been reached due to limitations of the single detection methods previously employed. Here, we aim to reach a broad understanding of changes in metabolite content using a genome-wide association study (GWAS) with eigenvector decomposition (EigenGWAS) on tomato accessions. RESULTS: An EigenGWAS was performed on 331 tomato accessions using the first eigenvector generated from the genomic data as a "phenotype" to understand the changes in fruit metabolite content during breeding. Two independent gene sets were identified that affected fruit metabolites during domestication and improvement in consumer-preferred tomatoes. Furthermore, 57 candidate genes related to polyphenol and polyamine biosynthesis were discovered, and a major candidate gene chlorogenate: glucarate caffeoyltransferase (SlCGT) was identified, which affected the quality and diseases resistance of tomato fruit, revealing the domestication mechanism of polyphenols. CONCLUSIONS: We identified gene sets that contributed to consumer liking during domestication and improvement of tomato. Our study reports novel evidence of selective sweeps and key metabolites controlled by multiple genes, increasing our understanding of the mechanisms of metabolites variation during those processes. It also supports a polygenic selection model for the application of tomato breeding.

The tomato OST1-VOZ1 module regulates drought-mediated flowering.

Flowering is a critical agricultural trait that substantially affects tomato fruit yield. Although drought stress influences flowering time, the molecular mechanism underlying drought-regulated flowering in tomato remains elusive. In this study, we demonstrated that loss of function of tomato OPEN STOMATA 1 (SlOST1), a protein kinase essential for abscisic acid (ABA) signaling and abiotic stress responses, lowers the tolerance of tomato plants to drought stress. slost1 mutants also exhibited a late flowering phenotype under both normal and drought stress conditions. We also established that SlOST1 directly interacts with and phosphorylates the NAC (NAM, ATAF and CUC)-type transcription factor VASCULAR PLANT ONE-ZINC FINGER 1 (SlVOZ1), at residue serine 67, thereby enhancing its stability and nuclear translocation in an ABA-dependent manner. Moreover, we uncovered several SlVOZ1 binding motifs from DNA affinity purification sequencing analyses and revealed that SlVOZ1 can directly bind to the promoter of the major flowering-integrator gene SINGLE FLOWER TRUSS to promote tomato flowering transition in response to drought. Collectively, our data uncover the essential role of the SlOST1-SlVOZ1 module in regulating flowering in response to drought stress in tomato and offer insights into a novel strategy to balance drought stress response and flowering.

Beyond stress response: OST1 opening doors for plants to grow.

The sucrose non-fermenting 1 (SNF1)-related protein kinase 2 (SnRK2) family members have been discovered to regulate abiotic stress response via the abscisic acid (ABA)-independent and dependent signaling pathways. SnRK2.6, also known as Open Stomata 1 (OST1), is a serine/threonine protein kinase that plays critical roles in linking ABA receptor complexes and downstream components such as transcription factors and anion channels to regulate stress response. Asides from its well-known regulatory roles in stomatal movement and cold stress response, OST1 has also been demonstrated recently to modulate major developmental roles of flowering and growth in plants. In this review, we will discuss about the various roles of OST1 as well as the 'doors' that OST1 can 'open' to help plants perform stress adaptation. Therefore, we will address how OST1 can regulate stomata apertures, cold stress tolerance as well as other aspects of its emerging roles such as balancing flowering and root growth in response to drought.

ZmERF21 directly regulates hormone signaling and stress-responsive gene expression to influence drought tolerance in maize seedlings.

Drought stress adversely impacts crop development and yield. Maize frequently encounters drought stress during its life cycle. Improvement of drought tolerance is a priority of maize breeding programs. Here, we identified a novel transcription factor encoding gene, APETALA2 (AP2)/Ethylene response factor (ERF), which is tightly associated with drought tolerance in maize seedlings. ZmERF21 is mainly expressed in the root and leaf and it can be highly induced by polyethylene glycol treatment. Genetic analysis showed that the zmerf21 mutant plants displayed a reduced drought tolerance phenotype, accompanied by phenotypical and physiological changes that are commonly observed in drought conditions. Overexpression of ZmERF21 in maize significantly increased the chlorophyll content and activities of antioxidant enzymes under drought conditions. RNA-Seq and DNA affinity purification sequencing analysis further revealed that ZmERF21 may directly regulate the expression of genes related to hormone (ethylene, abscisic acid) and Ca signaling as well as other stress-response genes through binding to the promoters of potential target genes. Our results thereby provided molecular evidence of ZmERF21 is involved in the drought stress response of maize.

A high-continuity and annotated tomato reference genome.

BACKGROUND: Genetic and functional genomics studies require a high-quality genome assembly. Tomato (Solanum lycopersicum), an important horticultural crop, is an ideal model species for the study of fruit development. RESULTS: Here, we assembled an updated reference genome of S. lycopersicum cv. Heinz 1706 that was 799.09 Mb in length, containing 34,384 predicted protein-coding genes and 65.66% repetitive sequences. By comparing the genomes of S. lycopersicum and S. pimpinellifolium LA2093, we found a large number of genomic fragments probably associated with human selection, which may have had crucial roles in the domestication of tomato. We also used a recombinant inbred line (RIL) population to generate a high-density genetic map with high resolution and accuracy. Using these resources, we identified a number of candidate genes that were likely to be related to important agronomic traits in tomato. CONCLUSION: Our results offer opportunities for understanding the evolution of the tomato genome and will facilitate the study of genetic mechanisms in tomato biology.

The blue light receptor CRY1 interacts with GID1 and DELLA proteins to repress gibberellin signaling and plant growth.

Improvements in plant architecture, such as reduced plant height under high-density planting, are important for agricultural production. Light and gibberellin (GA) are essential external and internal cues that affect plant architecture. In this study, we characterize the direct interaction of distinct receptors that link light and GA signaling in Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum L.). We show that the light receptor CRY1 represses GA signaling through interaction with all five DELLA proteins and promotion of RGA protein accumulation in Arabidopsis. Genetic analysis shows that CRY1-mediated growth repression is achieved by means of the DELLA proteins. Interestingly, we find that CRY1 also directly interacts with the GA receptor GID1 to competitively inhibit the GID1-GAI interaction. We also show that overexpression of TaCRY1a reduces plant height and coleoptile growth in wheat and that TaCRY1a interacts with both TaGID1 and Rht1 to competitively attenuate the TaGID1-Rht1 interaction. Based on these findings, we propose that the photoreceptor CRY1 competitively inhibits the GID1-DELLA interaction, thereby stabilizing DELLA proteins and enhancing their repression of plant growth.

The LRXs-RALFs-FER module controls plant growth and salt stress responses by modulating multiple plant hormones.

Salt stress is a major environmental factor limiting plant growth and productivity. We recently discovered an important new salt tolerance pathway, where the cell wall leucine-rich repeat extensins LRX3/4/5, the RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23 and receptor-like kinase FERONIA (FER) function as a module to simultaneously regulate plant growth and salt stress tolerance. However, the intracellular signaling pathways that are regulated by the extracellular LRX3/4/5-RALF22/23-FER module to coordinate growth, cell wall integrity and salt stress responses are still unknown. Here, we report that the LRX3/4/5-RALF22/23-FER module negatively regulates the levels of jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA). Blocking JA pathway rescues the dwarf phenotype of the lrx345 and fer-4 mutants, while disruption of ABA biosynthesis suppresses the salt-hypersensitivity of these mutants. Many salt stress-responsive genes display abnormal expression patterns in the lrx345 and fer-4 mutants, as well as in the wild type plants treated with epigallocatechin gallate (EGCG), an inhibitor of pectin methylesterases, suggesting cell wall integrity as a critical factor that determines the expression pattern of stress-responsive genes. Production of reactive oxygen species (ROS) is constitutively increased in the lrx345 and fer-4 mutants, and inhibition of ROS accumulation suppresses the salt-hypersensitivity of these mutants. Together, our work provides strong evidence that the LRX3/4/5-RALF22/23-FER module controls plant growth and salt stress responses by regulating hormonal homeostasis and ROS accumulation.

MIR156-Targeted SPL9 Is Phosphorylated by SnRK2s and Interacts With ABI5 to Enhance ABA Responses in Arabidopsis.

The miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors play key roles in regulating plant development, but little is known about their function in abscisic acid (ABA) signaling. Here, we report that the miR156-targeted SPLs enhance ABA responses and contribute to the inhibition of pre-harvest sprouting. We find that SPL9 directly activates the expression of ABA responsive genes through binding to their promoters. SPL9 was further shown to physically interact with ABSCISIC ACID INSENSITIVE 5 (ABI5), a master transcription factor in ABA signaling, thus promoting its association with the promoters of ABA responsive genes. Furthermore, we reveal that the protein kinases SnRK2s interact with and phosphorylate SPL9, which is essential for its role in the activation of ABA responses. Together, our results disclose a SnRK2s-SPLs-ABI5 regulatory module in ABA signaling in Arabidopsis.

Genome-wide DNA mutations in Arabidopsis plants after multigenerational exposure to high temperatures.

BACKGROUND: Elevated temperatures can cause physiological, biochemical, and molecular responses in plants that can greatly affect their growth and development. Mutations are the most fundamental force driving biological evolution. However, how long-term elevations in temperature influence the accumulation of mutations in plants remains unknown. RESULTS: Multigenerational exposure of Arabidopsis MA (mutation accumulation) lines and MA populations to extreme heat and moderate warming results in significantly increased mutation rates in single-nucleotide variants (SNVs) and small indels. We observe distinctive mutational spectra under extreme and moderately elevated temperatures, with significant increases in transition and transversion frequencies. Mutation occurs more frequently in intergenic regions, coding regions, and transposable elements in plants grown under elevated temperatures. At elevated temperatures, more mutations accumulate in genes associated with defense responses, DNA repair, and signaling. Notably, the distribution patterns of mutations among all progeny differ between MA populations and MA lines, suggesting that stronger selection effects occurred in populations. Methylation is observed more frequently at mutation sites, indicating its contribution to the mutation process at elevated temperatures. Mutations occurring within the same genome under elevated temperatures are significantly biased toward low gene density regions, special trinucleotides, tandem repeats, and adjacent simple repeats. Additionally, mutations found in all progeny overlap significantly with genetic variations reported in 1001 Genomes, suggesting non-uniform distribution of de novo mutations through the genome. CONCLUSION: Collectively, our results suggest that elevated temperatures can accelerate the accumulation, and alter the molecular profiles, of DNA mutations in plants, thus providing significant insight into how environmental temperatures fuel plant evolution.

The genome of Medicago polymorpha provides insights into its edibility and nutritional value as a vegetable and forage legume.

Medicago polymorpha is a nutritious and palatable forage and vegetable plant that also fixes nitrogen. Here, we reveal the chromosome-scale genome sequence of M. polymorpha using an integrated approach including Illumina, PacBio and Hi-C technologies. We combined PacBio full-length RNA-seq, metabolomic analysis, structural anatomy analysis and related physiological indexes to elucidate the important agronomic traits of M. polymorpha for forage and vegetable usage. The assembled M. polymorpha genome consisted of 457.53 Mb with a long scaffold N50 of 57.72 Mb, and 92.92% (441.83 Mb) of the assembly was assigned to seven pseudochromosomes. Comparative genomic analysis revealed that expansion and contraction of the photosynthesis and lignin biosynthetic gene families, respectively, led to enhancement of nutritious compounds and reduced lignin biosynthesis in M. polymorpha. In addition, we found that several positively selected nitrogen metabolism-related genes were responsible for crude protein biosynthesis. Notably, the metabolomic results revealed that a large number of flavonoids, vitamins, alkaloids, and terpenoids were enriched in M. polymorpha. These results imply that the decreased lignin content but relatively high nutrient content of M. polymorpha enhance its edibility and nutritional value as a forage and vegetable. Our genomic data provide a genetic basis that will accelerate functional genomic and breeding research on M. polymorpha as well as other Medicago and legume plants.