A G-type lectin receptor kinase negatively regulates Arabidopsis immunity against root-knot nematodes.

Root-knot nematodes (Meloidogyne spp., RKN) are responsible for extensive crop losses worldwide. During infection, they penetrate plant roots, migrate between plant cells, and establish feeding sites, known as giant cells, near the root vasculature. Previously, we found that nematode perception and early responses in plants were similar to those of microbial pathogens and required the BRI1-ASSOCIATED KINASE1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 (BAK1/SERK3) coreceptor in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum). Here, we implemented a reverse genetic screen for resistance or sensitivity to RKN using Arabidopsis T-DNA alleles of genes encoding transmembrane receptor-like kinases to identify additional receptors involved in this process. This screen identified a pair of allelic mutations with enhanced resistance to RKN in a gene we named ENHANCED RESISTANCE TO NEMATODES1 (ERN1). ERN1 encodes a G-type lectin receptor kinase (G-LecRK) with a single-pass transmembrane domain. Further characterization showed that ern1 mutants displayed stronger activation of MAP kinases, elevated levels of the defense marker MYB51, and enhanced H2O2 accumulation in roots upon RKN elicitor treatments. Elevated MYB51 expression and ROS bursts were also observed in leaves of ern1 mutants upon flg22 treatment. Complementation of ern1.1 with 35S- or native promoter-driven ERN1 rescued the RKN infection and enhanced defense phenotypes. Our results indicate that ERN1 is an important negative regulator of immunity.

Transcriptome analysis of aphid-resistant and susceptible near isogenic lines reveals candidate resistance genes in cowpea (Vigna unguiculata).

BACKGROUND: Cowpea (Vigna unguiculata) is a crucial crop for regions of the world that are prone to both heat and drought; however, the phytotoxic cowpea aphid (Aphis craccivora) impairs plant physiology at low population levels. Both antibiotic and antixenotic forms of resistance to the aphid have been mapped to two quantitative trait loci (QTLs) and near isogenic lines (NILs). The molecular mechanism for this resistance response remains unknown. RESULTS: To understand the genes underlying susceptibility and resistance, two cowpea lines with shared heritage were infested along a time course and characterized for transcriptome variation. Aphids remodeled cowpea development and signaling relative to host plant resistance and the duration of feeding, with resource acquisition and mobilization determining, in part, susceptibility to aphid attack. Major differences between the susceptible and resistant cowpea were identified including two regions of interest housing the most genetic differences between the lines. Candidate genes enabling aphid resistance include both conventional resistance genes (e.g., leucine rich repeat protein kinases) as well as multiple novel genes with no known orthologues. CONCLUSIONS: Our results demonstrate that feeding by the cowpea aphid globally remodels the transcriptome of cowpea, but how this occurs depends on both the duration of feeding and host-plant resistance. Constitutive expression profiles of the resistant genotype link aphid resistance to a finely-tuned resource management strategy that ultimately reduces damage (e.g., chlorosis) and delays cell turnover, while impeding aphid performance. Thus, aphid resistance in cowpea is a complex, multigene response that involves crosstalk between primary and secondary metabolism.

Non-canonical nematode endogenous retroviruses resulting from RNA virus glycoprotein gene capture by a metavirus.

Reverse-transcribing retroviruses exist as horizontally transmitted infectious agents or vertically transmitted endogenous retroviruses (ERVs) resident in eukaryotic genomes, and they are phylogenetically related to the long terminal repeat (LTR) class of retrotransposons. ERVs and retrotransposons are often distinguished only by the presence or absence of a gene encoding the envelope glycoprotein (env). Endogenous elements of the virus family Metaviridae include the insect-restricted Errantivirus genus of ERVs, for which some members possess env, and the pan-eukaryotic Metavirus genus that lacks an envelope glycoprotein gene. Here we report a novel Nematoda endogenous retrovirus (NERV) clade with core retroviral genes arranged uniquely as a continuous gag-env-pro-pol ORF. Reverse transcriptase sequences were phylogenetically related to metaviruses, but envelope glycoprotein sequences resembled those of the Nyamiviridae and Chrysoviridae RNA virus families, suggesting env gene capture during host cell infection by an RNA virus. NERVs were monophyletic, restricted to the nematode subclass Chromadoria, and included additional ORFs for a small hypothetical protein or a large Upf1-like RNA-dependent AAA-ATPase/helicase indicative of viral transduction of a host gene. Provirus LTR identity, low copy number, ORF integrity and segregation of three loci in Meloidogyne incognita, taken together with detection of NERV transcriptional activity, support potential infectivity of NERVs, along with their recent emergence and integration. Altogether, NERVs constitute a new and distinct Metaviridae lineage demonstrating retroviral evolution through sequential heterologous gene capture events.

Aphid effector Me10 interacts with tomato TFT7, a 14-3-3 isoform involved in aphid resistance.

We demonstrated previously that expression of Macrosiphum euphorbiae salivary protein Me10 enhanced aphid reproduction on its host tomato (Solanum lycopersicum). However, the mechanism of action of Me10 remained elusive. To confirm the secretion of Me10 by the aphid into plant tissues, we produced Me10 polyclonal antibodies. To identify the plant targets of Me10, we developed a tomato immune induced complementary DNA yeast two-hybrid library and screened it with Me10 as bait. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays were performed to validate one of the interactions in planta, and virus-induced gene silencing was used for functional characterization in tomato. We demonstrated that Me10 is secreted into the plant tissues and interacts with tomato 14-3-3 isoform 7 (TFT7) in yeast. Immunoprecipitation assays confirmed that Me10 and its homologue in Aphis gossypii, Ag10k, interact with TFT7 in planta. Further, BiFC revealed that Me10 interaction with TFT7 occurs in the plant cell cytoplasm. While silencing of TFT7 in tomato leaves did not affect tomato susceptibility to M. euphorbiae, it enhanced longevity and fecundity of A. gossypii, the non-host aphid. Our results suggest the model whereby TFT7 plays a role in aphid resistance in tomato and effectors of the Me10/Ag10k family interfere with TFT7 function during aphid infestation.

Advances in Plant-Nematode Interactions with Emphasis on the Notorious Nematode Genus Meloidogyne.

Plant infections by plant-parasitic nematodes (PPNs) continue to be one of the major limitations in agricultural systems. Root-knot nematodes (RKNs), belonging to the genus Meloidogyne, are one of the most important groups of PPNs worldwide. Their wide host range combined with ubiquitous presence, continues to provide challenges for their control and breeding for resistance. Although resistance to RKNs has been identified, incorporation of these resistances into crops and durability of the resistance remains challenging. In addition, progress in cloning of RKN resistance genes has been dismal. Recent identification of pattern-triggered immunity in roots against nematodes, an ascaroside as a nematode-associated molecular pattern (NAMP) and the discovery of a NAMP plant receptor, provide tools and opportunities to develop durable host resistance against nematodes including RKNs.

Classification and phylogenetic analyses of the Arabidopsis and tomato G-type lectin receptor kinases.

BACKGROUND: Pathogen perception by plants is mediated by plasma membrane-localized immune receptors that have varied extracellular domains. Lectin receptor kinases (LecRKs) are among these receptors and are subdivided into 3 classes, C-type LecRKs (C-LecRKs), L-type LecRKs (L-LecRKs) and G-type LecRKs (G-LecRKs). While C-LecRKs are represented by one or two members in all plant species investigated and have unknown functions, L-LecRKs have been characterized in a few plant species and have been shown to play roles in plant defense against pathogens. Whereas Arabidopsis G-LecRKs have been characterized, this family of LecRKs has not been studied in tomato. RESULTS: This investigation updates the current characterization of Arabidopsis G-LecRKs and characterizes the tomato G-LecRKs, using LecRKs from the monocot rice and the basal eudicot columbine to establish a basis for comparisons between the two core eudicots. Additionally, revisiting parameters established for Arabidopsis nomenclature for LecRKs is suggested for both Arabidopsis and tomato. Moreover, using phylogenetic analysis, we show the relationship among and between members of G-LecRKs from all three eudicot plant species. Furthermore, investigating presence of motifs in G-LecRKs we identified conserved motifs among members of G-LecRKs in tomato and Arabidopsis, with five present in at least 30 of the 38 Arabidopsis members and in at least 45 of the 73 tomato members. CONCLUSIONS: This work characterized tomato G-LecRKs and added members to the currently characterized Arabidopsis G-LecRKs. Additionally, protein sequence analysis showed an expansion of this family in tomato as compared to Arabidopsis, and the existence of conserved common motifs in the two plant species as well as conserved species-specific motifs.

Foundational and Translational Research Opportunities to Improve Plant Health.

Reader Comments | Submit a Comment The white paper reports the deliberations of a workshop focused on biotic challenges to plant health held in Washington, D.C. in September 2016. Ensuring health of food plants is critical to maintaining the quality and productivity of crops and for sustenance of the rapidly growing human population. There is a close linkage between food security and societal stability; however, global food security is threatened by the vulnerability of our agricultural systems to numerous pests, pathogens, weeds, and environmental stresses. These threats are aggravated by climate change, the globalization of agriculture, and an over-reliance on nonsustainable inputs. New analytical and computational technologies are providing unprecedented resolution at a variety of molecular, cellular, organismal, and population scales for crop plants as well as pathogens, pests, beneficial microbes, and weeds. It is now possible to both characterize useful or deleterious variation as well as precisely manipulate it. Data-driven, informed decisions based on knowledge of the variation of biotic challenges and of natural and synthetic variation in crop plants will enable deployment of durable interventions throughout the world. These should be integral, dynamic components of agricultural strategies for sustainable agriculture.

The Conformation of a Plasma Membrane-Localized Somatic Embryogenesis Receptor Kinase Complex Is Altered by a Potato Aphid-Derived Effector.

Somatic embryogenesis receptor kinases (SERKs) are transmembrane receptors involved in plant immunity. Tomato (Solanum lycopersicum) carries three SERK members. One of these, SlSERK1, is required for Mi-1.2-mediated resistance to potato aphids (Macrosiphum euphorbiae). Mi-1.2 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that in addition to potato aphids confers resistance to two additional phloem-feeding insects and to root-knot nematodes (Meloidogyne spp.). How SlSERK1 participates in Mi-1.2-mediated resistance is unknown, and no Mi-1.2 cognate pest effectors have been identified. Here, we study the mechanistic involvement of SlSERK1 in Mi-1.2-mediated resistance. We show that potato aphid saliva and protein extracts induce the Mi-1.2 defense marker gene SlWRKY72b, indicating that both saliva and extracts contain a Mi-1.2 recognized effector. Resistant tomato cultivar Motelle (Mi-1.2/Mi-1.2) plants overexpressing SlSERK1 were found to display enhanced resistance to potato aphids. Confocal microscopy revealed that Mi-1.2 localizes at three distinct subcellular compartments: the plasma membrane, cytoplasm, and nucleus. Coimmunoprecipitation experiments in these tomato plants and in Nicotiana benthamiana transiently expressing Mi-1.2 and SlSERK1 showed that Mi-1.2 and SlSERK1 colocalize only in a microsomal complex. Interestingly, bimolecular fluorescence complementation analysis showed that the interaction of Mi-1.2 and SlSERK1 at the plasma membrane distinctively changes in the presence of potato aphid saliva, suggesting a model in which a constitutive complex at the plasma membrane participates in defense signaling upon effector binding.

The Synthetic Elicitor 2-(5-Bromo-2-Hydroxy-Phenyl)-Thiazolidine-4-Carboxylic Acid Links Plant Immunity to Hormesis.

Synthetic elicitors are drug-like compounds that induce plant immune responses but are structurally distinct from natural defense elicitors. Using high-throughput screening, we previously identified 114 synthetic elicitors that activate the expression of a pathogen-responsive reporter gene in Arabidopsis (Arabidopsis thaliana). Here, we report on the characterization of one of these compounds, 2-(5-bromo-2-hydroxy-phenyl)-thiazolidine-4-carboxylic acid (BHTC). BHTC induces disease resistance of plants against bacterial, oomycete, and fungal pathogens and has a unique mode of action and structure. Surprisingly, we found that low doses of BHTC enhanced root growth in Arabidopsis, while high doses of this compound inhibited root growth, besides inducing defense. These effects are reminiscent of the hormetic response, which is characterized by low-dose stimulatory effects of a wide range of agents that are toxic or inhibitory at higher doses. Like its effects on defense, BHTC-induced hormesis in Arabidopsis roots is partially dependent on the WRKY70 transcription factor. Interestingly, BHTC-induced root hormesis is also affected in the auxin-response mutants axr1-3 and slr-1. By messenger RNA sequencing, we uncovered a dramatic difference between transcriptional profiles triggered by low and high doses of BHTC. Only high levels of BHTC induce typical defense-related transcriptional changes. Instead, low BHTC levels trigger a coordinated intercompartmental transcriptional response manifested in the suppression of photosynthesis- and respiration-related genes in the nucleus, chloroplasts, and mitochondria as well as the induction of development-related nuclear genes. Taken together, our functional characterization of BHTC links defense regulation to hormesis and provides a hypothetical transcriptional scenario for the induction of hormetic root growth.

GroEL from the endosymbiont Buchnera aphidicola betrays the aphid by triggering plant defense.

Aphids are sap-feeding plant pests and harbor the endosymbiont Buchnera aphidicola, which is essential for their fecundity and survival. During plant penetration and feeding, aphids secrete saliva that contains proteins predicted to alter plant defenses and metabolism. Plants recognize microbe-associated molecular patterns and induce pattern-triggered immunity (PTI). No aphid-associated molecular pattern has yet been identified. By mass spectrometry, we identified in saliva from potato aphids (Macrosiphum euphorbiae) 105 proteins, some of which originated from Buchnera, including the chaperonin GroEL. Because GroEL is a widely conserved bacterial protein with an essential function, we tested its role in PTI. Applying or infiltrating GroEL onto Arabidopsis (Arabidopsis thaliana) leaves induced oxidative burst and expression of PTI early marker genes. These GroEL-induced defense responses required the known coreceptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1. In addition, in transgenic Arabidopsis plants, inducible expression of groEL activated PTI marker gene expression. Moreover, Arabidopsis plants expressing groEL displayed reduced fecundity of the green peach aphid (Myzus persicae), indicating enhanced resistance against aphids. Furthermore, delivery of GroEL into tomato (Solanum lycopersicum) or Arabidopsis through Pseudomonas fluorescens, engineered to express the type III secretion system, also reduced potato aphid and green peach aphid fecundity, respectively. Collectively our data indicate that GroEL is a molecular pattern that triggers PTI.

A novel virus from Macrosiphum euphorbiae with similarities to members of the family Flaviviridae.

A virus with a large genome was identified in the transcriptome of the potato aphid (Macrosiphum euphorbiae) and was named Macrosiphum euphorbiae virus 1 (MeV-1). The MeV-1 genome is 22 780 nt in size, including 3' and 5' non-coding regions, with a single large ORF encoding a putative polyprotein of 7333 aa. The C-terminal region of the predicted MeV-1 polyprotein contained sequences with similarities to helicase, methyltransferase and RNA-dependent RNA polymerase (RdRp) motifs, while the N-terminal region lacked any motifs including structural proteins. Phylogenetic analysis of the helicase placed MeV-1 close to pestiviruses, while the RdRp region placed it close to pestiviruses and flaviviruses, suggesting MeV-1 has a positive-polarity ssRNA genome and is a member of the family Flaviviridae. Since the MeV-1 genome is predicted to contain a methyltransferase, a gene present typically in flaviviruses but not pestiviruses, MeV-1 is likely a member of the genus Flavivirus. MeV-1 was present in nymphal and adult stages of the aphid, aphid saliva and plant tissues fed upon by aphids. However, the virus was unable to multiply and spread in tomato plants. In addition, dsRNA, the replication intermediate of RNA viruses, was isolated from virus-infected M. euphorbiae and not from tomato plants infested with the aphid. Furthermore, nymphs laid without exposure to infected plants harboured the virus, indicating that MeV-1 is an aphid-infecting virus likely transmitted transovarially. The virus was present in M. euphorbiae populations from Europe but not from North America and was absent in all other aphid species tested.

MicroRNAs suppress NB domain genes in tomato that confer resistance to Fusarium oxysporum.

MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. We explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

Root-knot nematodes induce pattern-triggered immunity in Arabidopsis thaliana roots.

Root-knot nematodes (RKNs; Meloidogyne spp.) are plant parasites with a broad host range causing great losses worldwide. To parasitize their hosts, RKNs establish feeding sites in roots known as giant cells. The majority of work studying plant-RKN interactions in susceptible hosts addresses establishment of the giant cells and there is limited information on the early defense responses. Here we characterized early defense or pattern-triggered immunity (PTI) against RKNs in Arabidopsis thaliana. To address PTI, we evaluated known canonical PTI signaling mutants with RKNs and investigated the expression of PTI marker genes after RKN infection using both quantitative PCR and ß-glucuronidase reporter transgenic lines. We showed that PTI-compromised plants have enhanced susceptibility to RKNs, including the bak1-5 mutant. BAK1 is a common partner of distinct receptors of microbe- and damage-associated molecular patterns. Furthermore, our data indicated that nematode recognition leading to PTI responses involves camalexin and glucosinolate biosynthesis. While the RKN-induced glucosinolate biosynthetic pathway was BAK1-dependent, the camalexin biosynthetic pathway was only partially dependent on BAK1. Combined, our results indicate the presence of BAK1-dependent and -independent PTI against RKNs in A. thaliana, suggesting the existence of diverse nematode recognition mechanisms.

Hemipteran and dipteran pests: Effectors and plant host immune regulators.

Hemipteran and dipteran insects have behavioral, cellular and chemical strategies for evading or coping with the host plant defenses making these insects particularly destructive pests worldwide. A critical component of a host plant's defense to herbivory is innate immunity. Here we review the status of our understanding of the receptors that contribute to perception of hemipteran and dipteran pests and highlight the gaps in our knowledge in these early events in immune signaling. We also highlight recent advances in identification of the effectors that activate pattern-triggered immunity and those involved in effector-triggered immunity.

Potato aphid salivary proteome: enhanced salivation using resorcinol and identification of aphid phosphoproteins.

Aphids deliver saliva into plants and acquire plant sap for their nourishment using a specialized mouthpart or stylets. Aphid saliva is of great importance because it contains effectors that are involved in modulating host defense and metabolism. Although profiling aphid salivary glands and identifying secreted proteins have been successfully used, success in direct profiling of aphid saliva have been limited due to scarcity of saliva collected in artificial diets. Here we present the use of a neurostimulant, resorcinol, for inducing aphid salivation. Saliva of potato aphids (Macrosiphum euphorbiae), maintained on tomato, was collected in resorcinol diet. Salivary proteins were identified using mass spectrometry and compared with the existing M. euphorbiae salivary proteome collected in water. Comparative analysis was also performed with existing salivary proteomes from additional aphid species. Most of the proteins identified in the resorcinol diet were also present in the water diet and represented proteins with a plethora of functions in addition to a large number of unknowns. About half of the salivary proteins were not predicted for secretion or had canonical secretion signal peptides. We also analyzed the phosphorylation states of M. euphorbiae salivary proteins and identified three known aphid effectors, Me_WB01635/Mp1, Me10/Mp58, and Me23 that carry phosphorylation marks. In addition to insect proteins, tomato host proteins were also identified in aphid saliva. Our results indicate that aphid saliva is complex and provides a rich resource for functional characterization of effectors.

In planta expression or delivery of potato aphid Macrosiphum euphorbiae effectors Me10 and Me23 enhances aphid fecundity.

The interactions between aphids and their host plants seem to be analogous to those of plant-microbial pathogens. Unlike microbial pathogen effectors, little is known about aphid effectors and their ability to interfere with host immunity. To date, only three functional aphid effectors have been reported. To identify potato aphid (Macrosiphum euphorbiae) effectors, we developed a salivary gland transcriptome using Illumina technology. We generated 85 million Illumina reads from salivary glands and assembled them into 646 contigs. Ab initio sequence analysis predicted secretion signal peptides in 24% of these sequences, suggesting that they might be secreted into the plant during aphid feeding. Eight of these candidate effectors with secretion signal peptides were functionally characterized using Agrobacterium tumefaciens-mediated transient overexpression in Nicotiana benthamiana. Two candidate effectors, Me10 and Me23, increased aphid fecundity, suggesting their ability to suppress N. benthamiana defenses. Five of these candidate effectors, including Me10 and Me23, were also analyzed in tomato by delivering them through the Pseudomonas syringae type three secretion system. In tomato, only Me10 increased aphid fecundity. This work identified two additional aphid effectors with ability to manipulate the host for their advantage.

Hsp90 Gene Is Required for Mi-1-Mediated Resistance of Tomato to the Whitefly Bemisia tabaci.

The Mi-1 gene of tomato (Solanum lycopersicum) confers resistance against some nematodes and insects, but the resistance mechanisms differ depending on the harmful organism, as a hypersensitive reaction (HR) occurs only in the case of nematodes. The gene Rme1 is required for Mi-1-mediated resistance to nematodes, aphids, and whiteflies, and several additional proteins also play a role in this resistance. Among them, the involvement of the chaperone HSP90 has been demonstrated in Mi-1-mediated resistance for aphids and nematodes, but not for whiteflies. In this work, we studied the implication of the Hsp90 gene in the Mi-1 resistance against the whitefly Bemisia tabaci by means of Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS). The silencing of the Hsp90 gene in tomato Motelle plants carrying the Mi-1 gene resulted in a decrease in resistance to whiteflies, as oviposition values were significantly higher than those on non-silenced plants. This decrease in resistance was equivalent to that caused by the silencing of the Mi-1 gene itself. Infiltration with the control TRV vector did not alter Mi-1 mediated resistance to B. tabaci. Similar to the Mi-1 gene, silencing of Hsp90-1 occurs partially, as silenced plants showed a significant but not complete suppression of gene expression. Thus, our results demonstrate the requirement of Hsp90 in the Mi-1-mediated resistance to B. tabaci and reinforce the hypothesis of a common model for this resistance to nematodes and insects.

The receptor-like kinase SlSERK1 is required for Mi-1-mediated resistance to potato aphids in tomato.

The plant receptor-like kinase somatic embryogenesis receptor kinase 3 (SERK3)/brassinosteroid insensitive 1-associated kinase 1 (BAK1) is required for pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Here we show that a distinct member of the SERK family, SERK1, is required for the full functioning of Mi-1, a nucleotide binding leucine-rich repeat (NB-LRR) resistance protein. Mi-1 confers resistance to Meloidogyne spp. (root-knot nematodes, RKNs) and three phloem-feeding insects, including Macrosiphum euphorbiae (potato aphid). SERK1 was identified in a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) screen in Nicotiana benthamiana. The screen was based on the suppression of a pest-independent hypersensitive response triggered by a constitutively active form of Mi-1, Mi-DS4. To assess the role of SERK1 in Mi-1-mediated resistance, Solanum lycopersicum (tomato) SlSERK genes were cloned. Three SlSERK members were identified with homologies to Arabidopsis AtSERK1 or AtSERK3/BAK1, and were named SlSERK1, SlSERK3A and SlSERK3B. SlSERK1 is ubiquitously expressed in tomato. Reducing SlSERK1 transcript levels in resistant plants, using gene-specific TRV-SERK1 VIGS, revealed a role for SlSERK1 in Mi-1-mediated resistance to potato aphids, but not to RKNs. In addition, Mi-1-dependent SlWRKY72 gene regulation was compromised in SlSERK1-silenced plants, placing SlSERK1 in the Mi-1 signaling pathway. Silencing SlSERK1 in a susceptible tomato background did not reduce the susceptibility to aphids, indicating that SlSERK1 is unlikely to be an essential virulence target. SlSERK1 is an active kinase, mainly localized at the plasma membrane. This work identifies a critical early component of Mi-1 signaling, and demonstrates a role for SlSERK1 in NB-LRR-mediated immunity.

SlWRKY70 is required for Mi-1-mediated resistance to aphids and nematodes in tomato.

Plant resistance (R) gene-mediated defense responses against biotic stresses include vast transcriptional reprogramming. In several plant-pathogen systems, members of the WRKY family of transcription factors have been demonstrated to act as both positive and negative regulators of plant defense transcriptional networks. To identify the possible roles of tomato (Solanum lycopersicum) WRKY transcription factors in defense mediated by the R gene Mi-1 against potato aphid, Macrosiphum euphorbiae, and root-knot nematode (RKN), Meloidogyne javanica, we used tobacco rattle virus (TRV)-based virus-induced gene silencing and transcriptionally suppressed SlWRKY70, a tomato ortholog of the Arabidopsis thaliana WRKY70 gene. Silencing SlWRKY70 attenuated Mi-1-mediated resistance against both potato aphid and RKN showing that SlWRKY70 is required for Mi-1 function. Furthermore, we found SlWRKY70 transcripts to be inducible in response to aphid infestation and RKN inoculation. Mi-1-mediated recognition of these pests modulates this transcriptional response. As previously described for AtWRKY70, we found SlWRKY70 transcript levels to be up-regulated by salicylic acid and suppressed by methyl jasmonate. This indicates that some aspects of WRKY70 regulation are conserved among distantly related eudicots.

SEED: efficient clustering of next-generation sequences.

MOTIVATION: Similarity clustering of next-generation sequences (NGS) is an important computational problem to study the population sizes of DNA/RNA molecules and to reduce the redundancies in NGS data. Currently, most sequence clustering algorithms are limited by their speed and scalability, and thus cannot handle data with tens of millions of reads. RESULTS: Here, we introduce SEED-an efficient algorithm for clustering very large NGS sets. It joins sequences into clusters that can differ by up to three mismatches and three overhanging residues from their virtual center. It is based on a modified spaced seed method, called block spaced seeds. Its clustering component operates on the hash tables by first identifying virtual center sequences and then finding all their neighboring sequences that meet the similarity parameters. SEED can cluster 100 million short read sequences in <4 h with a linear time and memory performance. When using SEED as a preprocessing tool on genome/transcriptome assembly data, it was able to reduce the time and memory requirements of the Velvet/Oasis assembler for the datasets used in this study by 60-85% and 21-41%, respectively. In addition, the assemblies contained longer contigs than non-preprocessed data as indicated by 12-27% larger N50 values. Compared with other clustering tools, SEED showed the best performance in generating clusters of NGS data similar to true cluster results with a 2- to 10-fold better time performance. While most of SEED's utilities fall into the preprocessing area of NGS data, our tests also demonstrate its efficiency as stand-alone tool for discovering clusters of small RNA sequences in NGS data from unsequenced organisms. AVAILABILITY: The SEED software can be downloaded for free from this site: http://manuals.bioinformatics.ucr.edu/home/seed. CONTACT: thomas.girke@ucr.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.