A Solanum lycopersicoides reference genome facilitates insights into tomato specialized metabolism and immunity.

Wild relatives of tomato are a valuable source of natural variation in tomato breeding, as many can be hybridized to the cultivated species (Solanum lycopersicum). Several, including Solanum lycopersicoides, have been crossed to S. lycopersicum for the development of ordered introgression lines (ILs), facilitating breeding for desirable traits. Despite the utility of these wild relatives and their associated ILs, few finished genome sequences have been produced to aid genetic and genomic studies. Here we report a chromosome-scale genome assembly for S. lycopersicoides LA2951, which contains 37 938 predicted protein-coding genes. With the aid of this genome assembly, we have precisely delimited the boundaries of the S. lycopersicoides introgressions in a set of S. lycopersicum cv. VF36 × LA2951 ILs. We demonstrate the usefulness of the LA2951 genome by identifying several quantitative trait loci for phenolics and carotenoids, including underlying candidate genes, and by investigating the genome organization and immunity-associated function of the clustered Pto gene family. In addition, syntenic analysis of R2R3MYB genes sheds light on the identity of the Aubergine locus underlying anthocyanin production. The genome sequence and IL map provide valuable resources for studying fruit nutrient/quality traits, pathogen resistance, and environmental stress tolerance. We present a new genome resource for the wild species S. lycopersicoides, which we use to shed light on the Aubergine locus responsible for anthocyanin production. We also provide IL boundary mappings, which facilitated identifying novel carotenoid quantitative trait loci of which one was likely driven by an uncharacterized lycopene ß-cyclase whose function we demonstrate.

Influence of Flagellin Polymorphisms, Gene Regulation, and Responsive Memory on the Motility of Xanthomonas Species That Cause Bacterial Spot Disease of Solanaceous Plants.

Increasingly, new evidence has demonstrated variability in the epitope regions of bacterial flagellin, including in regions harboring the microbe-associated molecular patterns flg22 and flgII-28 that are recognized by the pattern recognition receptors FLS2 and FLS3, respectively. Additionally, because bacterial motility is known to contribute to pathogen virulence and chemotaxis, reductions in or loss of motility can significantly reduce bacterial fitness. In this study, we determined that variations in flg22 and flgII-28 epitopes allow some but not all Xanthomonas spp. to evade both FLS2- and FLS3-mediated oxidative burst responses. We observed variation in the motility for many isolates, regardless of their flagellin sequence. Instead, we determined that past growth conditions may have a significant impact on the motility status of isolates, because we could minimize this variability by inducing motility using chemoattractant assays. Additionally, motility could be significantly suppressed under nutrient-limited conditions, and bacteria could "remember" its prior motility status after storage at ultracold temperatures. Finally, we observed larger bacterial populations of strains with flagellin variants predicted not to be recognized by either FLS2 or FLS3, suggesting that these bacteria can evade flagellin recognition in tomato plants. Although some flagellin variants may impart altered motility and differential recognition by the host immune system, external growth parameters and gene expression regulation appear to have more significant impacts on the motility phenotypes for these Xanthomonas spp.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genome Sequencing and Functional Characterization of Xanthomonas cucurbitae, the Causal Agent of Bacterial Spot Disease of Cucurbits.

Bacterial leaf spot disease caused by Xanthomonas cucurbitae has severely affected the pumpkin industries in the Midwestern region of United States, with the bacteria mainly infecting pumpkin leaves and fruits, and leading to significant yield losses. In this study, we utilized genomics and genetics approaches to elucidate X. cucurbitae molecular mechanisms of pathogenesis during interaction with its host. We generated the first reference-quality whole-genome sequence of the X. cucurbitae type isolate and compared with other Xanthomonas species, X. cucurbitae has a smaller genome size with fewer virulence-related genes. RNA-seq analysis of X. cucurbitae under plant-mimicking media conditions showed altered transcriptional responses, with upregulation of virulence genes and downregulation of cellular homeostasis genes. Additionally, characterization of key virulence genes using gene deletion methods revealed that both type II enzymes and type III effectors are necessary for X. cucurbitae to cause infection in the pumpkin host.

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2.

Plants mount defense responses by recognizing indicators of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin, from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), contains two MAMPs, flg22 and flgII-28, that are recognized by tomato (Solanum lycopersicum) receptors Flagellin sensing2 (Fls2) and Fls3, respectively, but to what degree each receptor contributes to immunity and whether they promote immune responses using the same molecular mechanisms are unknown. Here, we characterized CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants and found that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, we observed striking differences in certain host responses mediated by the two receptors. Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species and an increase in transcript abundance of 44 tomato genes, with two genes serving as specific reporters for the Fls3 pathway. Fls3 had greater in vitro kinase activity than Fls2 and could transphosphorylate a substrate. Using chimeric Fls2/Fls3 proteins, we found no evidence that a single receptor domain is responsible for the Fls3-sustained reactive oxygen species, suggesting involvement of multiple structural features or a nullified function of the chimeric construct. This work reveals differences in certain immunity outputs between Fls2 and Fls3, suggesting that they might use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.

Survey of Sensitivity to Fatty Acid-Amino Acid Conjugates in the Solanaceae.

Plants perceive insect herbivores via a sophisticated surveillance system that detects a range of alarm signals, including herbivore-associated molecular patterns (HAMPs). Fatty acid-amino acid conjugates (FACs) are HAMPs present in oral secretions (OS) of lepidopteran larvae that induce defense responses in many plant species. In contrast to eggplant (Solanum melongena), tomato (S. lycopersicum) does not respond to FACs present in OS from Manduca sexta (Lepidoptera). Since both plants are found in the same genus, we tested whether loss of sensitivity to FACs in tomato may be a domestication effect. Using highly sensitive MAP kinase (MAPK) phosphorylation assays, we demonstrate that four wild tomato species and the closely related potato (S. tuberosum) do not respond to the FACs N-linolenoyl-L-glutamine and N-linolenoyl-L-glutamic acid, excluding a domestication effect. Among other genera within the Solanaceae, we found that bell pepper (Capsicum annuum) is responsive to FACs, while there is a differential responsiveness to FACs among tobacco (Nicotiana) species, ranging from strong responsiveness in N. benthamiana to no responsiveness in N. knightiana. The Petunia lineage is one of the oldest lineages within the Solanaceae and P. hybrida was responsive to FACs. Collectively, we demonstrate that plant responsiveness to FACs does not follow simple phylogenetic relationships in the family Solanaceae. Instead, sensitivity to FACs is a dynamic ancestral trait present in monocots and eudicots that was repeatedly lost during the evolution of Solanaceae species. Although tomato is insensitive to FACs, we found that other unidentified factors in M. sexta OS induce defenses in tomato.

Mai1 Protein Acts Between Host Recognition of Pathogen Effectors and Mitogen-Activated Protein Kinase Signaling.

The molecular mechanisms acting between host recognition of pathogen effectors by nucleotide-binding leucine-rich repeat receptor (NLR) proteins and mitogen-activated protein kinase (MAPK) signaling cascades are unknown. MAPKKKα (M3Kα) activates MAPK signaling leading to programmed cell death (PCD) associated with NLR-triggered immunity. We identified a tomato M3Kα-interacting protein, SlMai1, that has 80% amino acid identity with Arabidopsis brassinosteroid kinase 1 (AtBsk1). SlMai1 has a protein kinase domain and a C-terminal tetratricopeptide repeat domain that interacts with the kinase domain of M3Kα. Virus-induced gene silencing of Mai1 homologs in Nicotiana benthamiana increased susceptibility to Pseudomonas syringae and compromised PCD induced by four NLR proteins. PCD was restored by expression of a synthetic SlMai1 gene that resists silencing. Expression of AtBsk1 did not restore PCD in Mai1-silenced plants, suggesting SlMai1 is functionally divergent from AtBsk1. PCD caused by overexpression of M3Kα or MKK2 was unaffected by Mai1 silencing, suggesting Mai1 acts upstream of these proteins. Coexpression of Mai1 with M3Kα in leaves enhanced MAPK phosphorylation and accelerated PCD. These findings suggest Mai1 is a molecular link acting between host recognition of pathogens and MAPK signaling.

Natural variation for unusual host responses and flagellin-mediated immunity against Pseudomonas syringae in genetically diverse tomato accessions.

The interaction between tomato and Pseudomonas syringae pv tomato (Pst) is a well-developed model for investigating the molecular basis of the plant immune system. There is extensive natural variation in Solanum lycopersicum (tomato) but it has not been fully leveraged to enhance our understanding of the tomato-Pst pathosystem. We screened 216 genetically diverse accessions of cultivated tomato and a wild tomato species for natural variation in their response to three strains of Pst. The host response to Pst was investigated using multiple Pst strains, tomato accessions with available genome sequences, reactive oxygen species (ROS) assays, reporter genes and bacterial population measurements. The screen uncovered a broad range of previously unseen host symptoms in response to Pst, and one of these, stem galls, was found to be simply inherited. The screen also identified tomato accessions that showed enhanced responses to flagellin in bacterial population assays and in ROS assays upon exposure to flagellin-derived peptides, flg22 and flgII-28. Reporter genes confirmed that the host responses were due primarily to pattern recognition receptor-triggered immunity. This study revealed extensive natural variation in tomato for susceptibility and resistance to Pst and will enable elucidation of the molecular mechanisms underlying these host responses.

The Ptr1 Locus of Solanum lycopersicoides Confers Resistance to Race 1 Strains of Pseudomonas syringae pv. tomato and to Ralstonia pseudosolanacearum by Recognizing the Type III Effectors AvrRpt2 and RipBN.

Race 1 strains of Pseudomonas syringae pv. tomato, which cause bacterial speck disease of tomato, are becoming increasingly common and no simply inherited genetic resistance to such strains is known. We discovered that a locus in Solanum lycopersicoides, termed Pseudomonas tomato race 1 (Ptr1), confers resistance to race 1 P. syringae pv. tomato strains by detecting the activity of type III effector AvrRpt2. In Arabidopsis, AvrRpt2 degrades the RIN4 protein, thereby activating RPS2-mediated immunity. Using site-directed mutagenesis of AvrRpt2, we found that, like RPS2, activation of Ptr1 requires AvrRpt2 proteolytic activity. Ptr1 also detected the activity of AvrRpt2 homologs from diverse bacteria, including one in Ralstonia pseudosolanacearum. The genome sequence of S. lycopersicoides revealed no RPS2 homolog in the Ptr1 region. Ptr1 could play an important role in controlling bacterial speck disease and its future cloning may shed light on an example of convergent evolution for recognition of a widespread type III effector.

MAP kinases associate with high molecular weight multiprotein complexes.

Plant responses to the environment and developmental processes are mediated by a complex signaling network. The Arabidopsis thaliana mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 and their orthologs in other plants are shared signal transducers that respond to many developmental and environmental signals and thus represent highly connected hubs in the cellular signaling network. In animals, specific MAPK signaling complexes are assembled which enable input-specific protein-protein interactions and thus specific signaling outcomes. In plants, not much is known about such signaling complexes. Here, we report that MPK3, MPK6, and MPK10 orthologs in tomato, tobacco, and Arabidopsis as well as tomato MAPK kinase 4 (MKK4) associate with high molecular weight (~250-550 kDa) multiprotein complexes. Elicitation by the defense-associated peptides flg22 and systemin resulted in phosphorylation and activation of the monomeric MAPKs, whereas the complex-associated MAPKs remained unphosphorylated and inactive. In contrast, treatment of tomato cells with a phosphatase inhibitor resulted in association of phosphorylated MPK1/2 with the complex. These results demonstrate that plant MAPKs and MAPKKs dynamically assemble into stable multiprotein complexes and this may depend on their phosphorylation status. Identification of the constituents of these multiprotein complexes promises a deeper understanding of signaling dynamics.

Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry.

BACKGROUND: The plant plasma membrane is a key battleground in the war between plants and their pathogens. Plants detect the presence of pathogens at the plasma membrane using sensor proteins, many of which are targeted to this lipophilic locale by way of fatty acid modifications. Pathogens secrete effector proteins into the plant cell to suppress the plant's defense mechanisms. These effectors are able to access and interfere with the surveillance machinery at the plant plasma membrane by hijacking the host's fatty acylation apparatus. Despite the important involvement of protein fatty acylation in both plant immunity and pathogen virulence mechanisms, relatively little is known about the role of this modification during plant-pathogen interactions. This dearth in our understanding is due largely to the lack of methods to monitor protein fatty acid modifications in the plant cell. RESULTS: We describe a rapid method to detect two major forms of fatty acylation, N-myristoylation and S-acylation, of candidate proteins using alkyne fatty acid analogs coupled with click chemistry. We applied our approach to confirm and decisively demonstrate that the archetypal pattern recognition receptor FLS2, the well-characterized pathogen effector AvrPto, and one of the best-studied intracellular resistance proteins, Pto, all undergo plant-mediated fatty acylation. In addition to providing a means to readily determine fatty acylation, particularly myristoylation, of candidate proteins, this method is amenable to a variety of expression systems. We demonstrate this using both Arabidopsis protoplasts and stable transgenic Arabidopsis plants and we leverage Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves as a means for high-throughput evaluation of candidate proteins. CONCLUSIONS: Protein fatty acylation is a targeting tactic employed by both plants and their pathogens. The metabolic labeling approach leveraging alkyne fatty acid analogs and click chemistry described here has the potential to provide mechanistic details of the molecular tactics used at the host plasma membrane in the battle between plants and pathogens.

Tomato receptor FLAGELLIN-SENSING 3 binds flgII-28 and activates the plant immune system.

Plants and animals detect the presence of potential pathogens through the perception of conserved microbial patterns by cell surface receptors. Certain solanaceous plants, including tomato, potato and pepper, detect flgII-28, a region of bacterial flagellin that is distinct from that perceived by the well-characterized FLAGELLIN-SENSING 2 receptor. Here we identify and characterize the receptor responsible for this recognition in tomato, called FLAGELLIN-SENSING 3. This receptor binds flgII-28 and enhances immune responses leading to a reduction in bacterial colonization of leaf tissues. Further characterization of FLS3 and its signalling pathway could provide new insights into the plant immune system and transfer of the receptor to other crop plants offers the potential of enhancing resistance to bacterial pathogens that have evolved to evade FLS2-mediated immunity.

Natural variation for responsiveness to flg22, flgII-28, and csp22 and Pseudomonas syringae pv. tomato in heirloom tomatoes.

Tomato (Solanum lycopersicum L.) is susceptible to many diseases including bacterial speck caused by Pseudomonas syringae pv. tomato. Bacterial speck disease is a serious problem worldwide in tomato production areas where moist conditions and cool temperatures occur. To enhance breeding of speck resistant fresh-market tomato cultivars we identified a race 0 field isolate, NC-C3, of P. s. pv. tomato in North Carolina and used it to screen a collection of heirloom tomato lines for speck resistance in the field. We observed statistically significant variation among the heirloom tomatoes for their response to P. s. pv. tomato NC-C3 with two lines showing resistance approaching a cultivar that expresses the Pto resistance gene, although none of the heirloom lines have Pto. Using an assay that measures microbe-associated molecular pattern (MAMP)-induced production of reactive oxygen species (ROS), we investigated whether the heirloom lines showed differential responsiveness to three bacterial-derived peptide MAMPs: flg22 and flgII-28 (from flagellin) and csp22 (from cold shock protein). Significant differences were observed for MAMP responsiveness among the lines, although these differences did not correlate strongly with resistance or susceptibility to bacterial speck disease. The identification of natural variation for MAMP responsiveness opens up the possibility of using a genetic approach to identify the underlying loci and to facilitate breeding of cultivars with enhanced disease resistance. Towards this goal, we discovered that responsiveness to csp22 segregates as a single locus in an F2 population of tomato.

Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility.

The bacterial flagellin (FliC) epitopes flg22 and flgII-28 are microbe-associated molecular patterns (MAMPs). Although flg22 is recognized by many plant species via the pattern recognition receptor FLS2, neither the flgII-28 receptor nor the extent of flgII-28 recognition by different plant families is known. Here, we tested the significance of flgII-28 as a MAMP and the importance of allelic diversity in flg22 and flgII-28 in plant-pathogen interactions using purified peptides and a Pseudomonas syringae ∆fliC mutant complemented with different fliC alleles. The plant genotype and allelic diversity in flg22 and flgII-28 were found to significantly affect the plant immune response, but not bacterial motility. The recognition of flgII-28 is restricted to a number of solanaceous species. Although the flgII-28 peptide does not trigger any immune response in Arabidopsis, mutations in both flg22 and flgII-28 have FLS2-dependent effects on virulence. However, the expression of a tomato allele of FLS2 does not confer to Nicotiana benthamiana the ability to detect flgII-28, and tomato plants silenced for FLS2 are not altered in flgII-28 recognition. Therefore, MAMP diversification is an effective pathogen virulence strategy, and flgII-28 appears to be perceived by an as yet unidentified receptor in the Solanaceae, although it has an FLS2-dependent virulence effect in Arabidopsis.

Wounding systemically activates a mitogen-activated protein kinase in forage and turf grasses.

Forage and turf grasses are continually cut and grazed by livestock, however very little is known concerning the perception or molecular responses to wounding. Mechanical wounding rapidly activated a 46 kDa and a 44 kDa mitogen-activated protein kinase (MAPK) in six different grass species. In the model grass species Lolium temulentum, the 46 kDa MAPK was rapidly activated within 5 min of wounding both locally and systemically in an adjacent unwounded tiller. This indicates that wounding generates a rapidly propagated long-distance signal that activates a MAPK in the distal portions of the plant. This 46 kDa MAPK activity was not enhanced by the addition of the pathogen-associated signal salicylic acid (SA) to the wound site nor induced when exposed to methyl jasmonate (MJ), which is a potent inducer of the wound response in dicotyledonous plants. However, pretreatment with MJ increased the wound-induced activity of the 44 kDa MAPK over the activity in control plants.

The COP9 signalosome controls jasmonic acid synthesis and plant responses to herbivory and pathogens.

The COP9 signalosome (CSN) is a multi-protein complex that regulates the activities of cullin-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate proteins in order to target them for proteasomal degradation. The CSN is required for proper plant development. Here we show that the CSN also has a profound effect on plant defense responses. Silencing of genes for CSN subunits in tomato plants resulted in a mild morphological phenotype and reduced expression of wound-responsive genes in response to mechanical wounding, attack by Manduca sexta larvae, and Prosystemin over-expression. In contrast, expression of pathogenesis-related genes was increased in a stimulus-independent manner in these plants. The reduced wound response in CSN-silenced plants corresponded with reduced synthesis of jasmonic acid (JA), but levels of salicylic acid (SA) were unaltered. As a consequence, these plants exhibited reduced resistance against herbivorous M. sexta larvae and the necrotrophic fungal pathogen Botrytis cinerea. In contrast, susceptibility to tobacco mosaic virus (TMV) was not altered in CSN-silenced plants. These data demonstrate that the CSN orchestrates not only plant development but also JA-dependent plant defense responses.

Tissue-type specific systemin perception and the elusive systemin receptor.

Systemin is a wound signaling peptide from tomato that is important for plant defenses against herbivory. The systemin receptor was initially identified as the tomato homolog of the brassinosteroid receptor BRI1, but genetic evidence argued against this finding. However, we found that BRI1 may function as an inappropriate systemin binding protein that does not activate the systemin signaling pathway. Here we provide evidence that systemin perception is localized in a tissue-type specific manner. Mesophyll protoplasts were not sensitive to systemin, while they responded to other elicitors. We hypothesize that the elusive systemin receptor is a protein with high similarity to BRI1 which is specifically localized in vascular tissue like the systemin precursor prosystemin. Binding of systemin to BRI1 may be an artifact of transgenic BRI1-overexpressing plants, but does not take place in wild type tomato cells.