Biological Control of Three Major Cucumber and Pepper Pests: Whiteflies, Thrips, and Spider Mites, in High Plastic Tunnels Using Two Local Phytoseiid Mites.

To enhance food security, food safety, and environmental health, a bio-based integrated pest management (BIPM) strategy was evaluated at two coastal locations in Lebanon as an alternative to toxic pesticide sprays in commercial high-arched plastic tunnels common in many countries. The evaluation occurred during two cucumber and pepper cropping seasons: spring and fall. At each site, two commercial tunnels were used; farmers' conventional practices were applied in one tunnel, while the BIPM approach was followed in the second tunnel. In the farmers' practices, a total of 14 sprays of insecticide/acaricide mixtures were applied during the spring growing season, and 6 sprays were applied during the fall. In the BIPM tunnels, hotspot releases of local strains of Amblyseius swirskii and Phytoseiulus persimilis were applied. By the end of the spring season, the number of whitefly nymphs (WFNs)/leaf and thrips/leaf in the pesticide treatment were 4.8 and 0.06, respectively, compared to 0.1 and 0.33, respectively, in the BIPM treatment. Similarly, at the end of the fall season, the WFNs reached 19.7/leaf in the pesticide control as compared to 1.2/leaf in the BIPM treatment, proving the efficacy of A. swirskii. Farmers using conventional acaricides during both cropping seasons failed to control Tetranychus urticae, the two-spotted spider mite (TSSM). However, hotspot releases of P. persimilis were successful in controlling TSSM. By the end of June, the number of TSSMs reached 7.8/leaf in the BIPM treatment compared to 53/leaf in the pesticide treatment. Likewise, in December, TSSM numbers reached 9/leaf in the BIPM treatment compared to 40/leaf in the pesticide treatment. Preliminary observations of pepper showed that both predatory mites (A. swirskii and P. persimilis) gave similar or better efficacy against the three pests. The two local predatory phytoseiid mites seem to be effective in controlling these three major pests and to be adapted to local environmental conditions. A rate of increase of 0.86 was observed for P. persimilis and 0.22 for A. swirskii, in June, when maximum temperatures were close to 40 °C. This also shows a compatibility between the two predators. In conclusion, our BIPM approach was efficient under a Mediterranean climate in arched plastic tunnels with relatively poor aeration.

Promoting student interest in plant biology through an inquiry-based module exploring plant circadian rhythm, gene expression, and defense against insects.

We present a weeklong curricular module for high school biology students that promotes knowledge of phytohormones, the circadian clock, and the Central Dogma. The module, which relies on easily accessible items and requires minimal space, integrates a hands-on experiment that guides students through replicating research examining circadian entrainment in postharvest cabbage from groceries. This work found that plants have cyclical, circadian expression of genes that produce phytohormones, and that such cyclical expression influences herbivory by caterpillars. Such cyclical patterns were found in plants both in situ and in postharvest cabbage. This work thus provides an ideal platform to shape student conceptions of circadian rhythms, gene expression, and plant herbivory by having students use light timers to entrain postharvest cabbage to alternating light and dark cycles and then measuring herbivory in these plants. The results should replicate previous work and demonstrate less herbivory when both plant and caterpillar are entrained to the same light and dark cycles since the expression of phytohormones involved in plant defense will be greatest when caterpillars are active. The module then concludes with a discussion of gene regulation and how this influences phytohormones. This module was field tested at four public schools, reaching over 600 students, and we present data demonstrating that the module led to learning gains and likely increases in interest in plant biology and self-efficacy.

De Novo Drug Design Using Transformer-Based Machine Translation and Reinforcement Learning of an Adaptive Monte Carlo Tree Search.

The discovery of novel therapeutic compounds through de novo drug design represents a critical challenge in the field of pharmaceutical research. Traditional drug discovery approaches are often resource intensive and time consuming, leading researchers to explore innovative methods that harness the power of deep learning and reinforcement learning techniques. Here, we introduce a novel drug design approach called drugAI that leverages the Encoder-Decoder Transformer architecture in tandem with Reinforcement Learning via a Monte Carlo Tree Search (RL-MCTS) to expedite the process of drug discovery while ensuring the production of valid small molecules with drug-like characteristics and strong binding affinities towards their targets. We successfully integrated the Encoder-Decoder Transformer architecture, which generates molecular structures (drugs) from scratch with the RL-MCTS, serving as a reinforcement learning framework. The RL-MCTS combines the exploitation and exploration capabilities of a Monte Carlo Tree Search with the machine translation of a transformer-based Encoder-Decoder model. This dynamic approach allows the model to iteratively refine its drug candidate generation process, ensuring that the generated molecules adhere to essential physicochemical and biological constraints and effectively bind to their targets. The results from drugAI showcase the effectiveness of the proposed approach across various benchmark datasets, demonstrating a significant improvement in both the validity and drug-likeness of the generated compounds, compared to two existing benchmark methods. Moreover, drugAI ensures that the generated molecules exhibit strong binding affinities to their respective targets. In summary, this research highlights the real-world applications of drugAI in drug discovery pipelines, potentially accelerating the identification of promising drug candidates for a wide range of diseases.

Multiple light signaling pathways control solar tracking in sunflowers.

Sunflowers are famous for their ability to track the sun throughout the day and then reorient at night to face east the following morning. This occurs by differential growth patterns, with the east sides of stems growing more during the day and the west sides of stems growing more at night. This process, termed heliotropism, is generally believed to be a specialized form of phototropism; however, the underlying mechanism is unknown. To better understand heliotropism, we compared gene expression patterns in plants undergoing phototropism in a controlled environment and in plants initiating and maintaining heliotropic growth in the field. We found the expected transcriptome signatures of phototropin-mediated phototropism in sunflower stems bending towards monochromatic blue light. Surprisingly, the expression patterns of these phototropism-regulated genes are quite different in heliotropic plants. Most genes rapidly induced during phototropism display only minor differences in expression across solar tracking stems. However, some genes that are both rapidly induced during phototropism and are implicated in growth responses to foliar shade are rapidly induced on the west sides of stems at the onset of heliotropism, suggesting a possible role for red light photoreceptors in solar tracking. To test the involvement of different photoreceptor signaling pathways in heliotropism, we modulated the light environment of plants initiating solar tracking. We found that depletion of either red and far-red light or blue light did not hinder the initiation or maintenance of heliotropism in the field. Together, our results suggest that the transcriptional regulation of heliotropism is distinct from phototropin-mediated phototropism and likely involves inputs from multiple light signaling pathways.

The Sw-5b NLR Immune Receptor Induces Early Transcriptional Changes in Response to Thrips and Mechanical Modes of Inoculation of Tomato spotted wilt orthotospovirus.

The NLR (nucleotide-binding leucine-rich repeat) class immune receptor Sw-5b confers resistance to Tomato spotted wilt orthotospovirus (TSWV). Although Sw-5b is known to activate immunity upon recognition of the TSWV movement protein NSm, we know very little about the downstream events that lead to resistance. Here, we investigated the Sw-5b-mediated early transcriptomic changes that occur in response to mechanical and thrips-mediated inoculation of TSWV, using near-isogenic tomato lines CNPH-LAM 147 (Sw5b+/+) and Santa Clara (Sw-5b-/-). We observed earlier Sw-5b-mediated transcriptional changes in response to thrips-mediated inoculation compared with that in response to mechanical inoculation of TSWV. With thrips-mediated inoculation, differentially expressed genes (DEGs) were observed at 12, 24, and 72 h postinoculation (hpi). Whereas with mechanical inoculation, DEGs were observed only at 72 hpi. Although some DEGs were shared between the two methods of inoculation, many DEGs were specific to either thrips-mediated or mechanical inoculation of TSWV. In response to thrips-mediated inoculation, an NLR immune receptor, cysteine-rich receptor-like kinase, G-type lectin S-receptor-like kinases, the ethylene response factor 1, and the calmodulin-binding protein 60 were induced. Fatty acid desaturase 2-9, cell death genes, DCL2b, RIPK/PBL14-like, ERF017, and WRKY75 were differentially expressed in response to mechanical inoculation. Our findings reveal Sw-5b responses specific to the method of TSWV inoculation. Although TSWV is transmitted in nature primarily by the thrips, Sw-5b responses to thrips inoculation have not been previously studied. Therefore, the DEGs we have identified in response to thrips-mediated inoculation provide a new foundation for understanding the mechanistic roles of these genes in the Sw-5b-mediated resistance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Virtual and In Vitro Screening of Natural Products Identifies Indole and Benzene Derivatives as Inhibitors of SARS-CoV-2 Main Protease (Mpro).

The rapid spread of the coronavirus disease 2019 (COVID-19) resulted in serious health, social, and economic consequences. While the development of effective vaccines substantially reduced the severity of symptoms and the associated deaths, we still urgently need effective drugs to further reduce the number of casualties associated with SARS-CoV-2 infections. Machine learning methods both improved and sped up all the different stages of the drug discovery processes by performing complex analyses with enormous datasets. Natural products (NPs) have been used for treating diseases and infections for thousands of years and represent a valuable resource for drug discovery when combined with the current computation advancements. Here, a dataset of 406,747 unique NPs was screened against the SARS-CoV-2 main protease (Mpro) crystal structure (6lu7) using a combination of ligand- and structural-based virtual screening. Based on 1) the predicted binding affinities of the NPs to the Mpro, 2) the types and number of interactions with the Mpro amino acids that are critical for its function, and 3) the desirable pharmacokinetic properties of the NPs, we identified the top 20 candidates that could potentially inhibit the Mpro protease function. A total of 7 of the 20 top candidates were subjected to in vitro protease inhibition assay and 4 of them (4/7; 57%), including two beta carbolines, one N-alkyl indole, and one Benzoic acid ester, had significant inhibitory activity against Mpro protease. These four NPs could be developed further for the treatment of COVID-19 symptoms.

Evaluation of qPCR to Detect Shifts in Population Composition of the Rhizobial Symbiont Mesorhizobium japonicum during Serial in Planta Transfers.

Microbial symbionts range from mutualistic to commensal to antagonistic. While these roles are distinct in their outcome, they are also fluid in a changing environment. Here, we used the Lotus japonicus-Mesorhizobium japonicum symbiosis to investigate short-term and long-term shifts in population abundance using an effective, fast, and low-cost tracking methodology for M. japonicum. We use quantitative polymerase chain reaction (qPCR) to track previously generated signature-tagged M. japonicum mutants targeting the Tn5 transposon insertion and the flanking gene. We used a highly beneficial wild type and moderately beneficial and non-beneficial mutants of M. japonicum sp. nov. to demonstrate the specificity of these primers to estimate the relative abundance of each genotype within individual nodules and after serial transfers to new hosts. For the moderate and non-beneficial genotypes, qPCR allowed us to differentiate genotypes that are phenotypically indistinguishable and investigate host control with suboptimal symbionts. We consistently found the wild type increasing in the proportion of the population, but our data suggest a potential reproductive trade-off between the moderate and non-beneficial genotypes. The multi-generation framework we used, coupled with qPCR, can easily be scaled up to track dozens of M. japonicum mutants simultaneously. Moreover, these mutants can be used to explore M. japonicum genotype abundance in the presence of a complex soil community.

De novo Sequencing and Analysis of Salvia hispanica Tissue-Specific Transcriptome and Identification of Genes Involved in Terpenoid Biosynthesis.

Salvia hispanica (commonly known as chia) is gaining popularity worldwide as a healthy food supplement due to its low saturated fatty acid and high polyunsaturated fatty acid content, in addition to being rich in protein, fiber, and antioxidants. Chia leaves contain plethora of secondary metabolites with medicinal properties. In this study, we sequenced chia leaf and root transcriptomes using the Illumina platform. The short reads were assembled into contigs using the Trinity software and annotated against the Uniprot database. The reads were de novo assembled into 103,367 contigs, which represented 92.8% transcriptome completeness and a diverse set of Gene Ontology terms. Differential expression analysis identified 6151 and 8116 contigs significantly upregulated in the leaf and root tissues, respectively. In addition, we identified 30 contigs belonging to the Terpene synthase (TPS) family and demonstrated their evolutionary relationships to tomato TPS family members. Finally, we characterized the expression of S. hispanica TPS members in leaves subjected to abiotic stresses and hormone treatments. Abscisic acid had the most pronounced effect on the expression of the TPS genes tested in this study. Our work provides valuable community resources for future studies aimed at improving and utilizing the beneficial constituents of this emerging healthy food source.

Sequence analysis of the potato aphid Macrosiphum euphorbiae transcriptome identified two new viruses.

The potato aphid, Macrosiphum euphorbiae, is an important agricultural pest that causes economic losses to potato and tomato production. To establish the transcriptome for this aphid, RNA-Seq libraries constructed from aphids maintained on tomato plants were used in Illumina sequencing generating 52.6 million 75-105 bp paired-end reads. The reads were assembled using Velvet/Oases software with SEED preprocessing resulting in 22,137 contigs with an N50 value of 2,003bp. After removal of contigs from tomato host origin, 20,254 contigs were annotated using BLASTx searches against the non-redundant protein database from the National Center for Biotechnology Information (NCBI) as well as IntereProScan. This identified matches for 74% of the potato aphid contigs. The highest ranking hits for over 12,700 contigs were against the related pea aphid, Acyrthosiphon pisum. Gene Ontology (GO) was used to classify the identified M. euphorbiae contigs into biological process, cellular component and molecular function. Among the contigs, sequences of microbial origin were identified. Sixty five contigs were from the aphid bacterial obligate endosymbiont Buchnera aphidicola origin and two contigs had amino acid similarities to viruses. The latter two were named Macrosiphum euphorbiae virus 2 (MeV-2) and Macrosiphum euphorbiae virus 3 (MeV-3). The highest sequence identity to MeV-2 had the Dysaphis plantaginea densovirus, while to MeV-3 is the Hubei sobemo-like virus 49. Characterization of MeV-2 and MeV-3 indicated that both are transmitted vertically from adult aphids to nymphs. MeV-2 peptides were detected in the aphid saliva and only MeV-2 and not MeV-3 nucleic acids were detected inside tomato leaves exposed to virus-infected aphids. However, MeV-2 nucleic acids did not persist in tomato leaf tissues, after clearing the plants from aphids, indicating that MeV-2 is likely an aphid virus.

Circadian regulation of sunflower heliotropism, floral orientation, and pollinator visits.

Young sunflower plants track the Sun from east to west during the day and then reorient during the night to face east in anticipation of dawn. In contrast, mature plants cease movement with their flower heads facing east. We show that circadian regulation of directional growth pathways accounts for both phenomena and leads to increased vegetative biomass and enhanced pollinator visits to flowers. Solar tracking movements are driven by antiphasic patterns of elongation on the east and west sides of the stem. Genes implicated in control of phototropic growth, but not clock genes, are differentially expressed on the opposite sides of solar tracking stems. Thus, interactions between environmental response pathways and the internal circadian oscillator coordinate physiological processes with predictable changes in the environment to influence growth and reproduction.

Circadian regulation of hormone signaling and plant physiology.

The survival and reproduction of plants depend on their ability to cope with a wide range of daily and seasonal environmental fluctuations during their life cycle. Phytohormones are plant growth regulators that are involved in almost every aspect of growth and development as well as plant adaptation to myriad abiotic and biotic conditions. The circadian clock, an endogenous and cell-autonomous biological timekeeper that produces rhythmic outputs with close to 24-h rhythms, provides an adaptive advantage by synchronizing plant physiological and metabolic processes to the external environment. The circadian clock regulates phytohormone biosynthesis and signaling pathways to generate daily rhythms in hormone activity that fine-tune a range of plant processes, enhancing adaptation to local conditions. This review explores our current understanding of the interplay between the circadian clock and hormone signaling pathways.

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.

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.

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.

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.

Construction of RNA-Seq libraries from large and microscopic tissues for the Illumina sequencing platform.

Second-generation DNA sequencing platforms have emerged as powerful tools in biological research. Their high sequence output at lower cost and minimal input DNA requirement render them suitable for broad applications ranging from gene expression studies to personalized clinical diagnostics. Here, we describe the preparation of cDNA libraries, from both whole aphid insects and their microscopic salivary gland tissues, suitable for high-throughput DNA sequencing on the Illumina platform.

High and low throughput screens with root-knot nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28°C. Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

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.

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.

WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-for-gene resistance mediated by the tomato R gene Mi-1.

WRKY transcription factors play a central role in transcriptional reprogramming associated with plant immune responses. However, due to functional redundancy, typically the contribution of individual members of this family to immunity is only subtle. Using microarray analysis, we found that the paralogous tomato WRKY genes SlWRKY72a and b are transcriptionally up-regulated during disease resistance mediated by the R gene Mi-1. Virus-induced gene silencing of these two genes in tomato resulted in a clear reduction of Mi-1-mediated resistance as well as basal defense against root-knot nematodes (RKN) and potato aphids. Using Arabidopsis T-DNA insertion mutants, we found that their Arabidopsis ortholog, AtWRKY72, is also required for full basal defense against RKN as well as to the oomycete Hyaloperonospora arabidopsidis. Despite their similar roles in basal defense against RKN in both tested plant species, WRKY72-type transcription factors in tomato, but not in Arabidopsis, clearly contributed to basal defense against the bacterial pathogen Pseudomonas syringae. Of the five R genes that we tested in tomato and Arabidopsis, only Mi-1 appeared to be dependent on WRKY72-type transcription factors. Interestingly, AtWRKY72 target genes, identified by microarray analysis of H. arabidopsidis-triggered transcriptional changes, appear to be largely non-responsive to analogs of the defense hormone salicylic acid (SA). Thus, similarly to Mi-1, which in part acts independently of SA, AtWRKY72 appears to utilize SA-independent defense mechanisms. We propose that WRKY72-type transcription factors play a partially conserved role in basal defense in tomato and Arabidopsis, a function that has been recruited to serve Mi-1-dependent immunity.