A butterfly egg-killing hypersensitive response in Brassica nigra is controlled by a single locus, PEK, containing a cluster of TIR-NBS-LRR receptor genes.

Knowledge of plant recognition of insects is largely limited to a few resistance (R) genes against sap-sucking insects. Hypersensitive response (HR) characterizes monogenic plant traits relying on R genes in several pathosystems. HR-like cell death can be triggered by eggs of cabbage white butterflies (Pieris spp.), pests of cabbage crops (Brassica spp.), reducing egg survival and representing an effective plant resistance trait before feeding damage occurs. Here, we performed genetic mapping of HR-like cell death induced by Pieris brassicae eggs in the black mustard Brassica nigra (B. nigra). We show that HR-like cell death segregates as a Mendelian trait and identified a single dominant locus on chromosome B3, named PEK (Pieris  egg- killing). Eleven genes are located in an approximately 50 kb region, including a cluster of genes encoding intracellular TIR-NBS-LRR (TNL) receptor proteins. The PEK locus is highly polymorphic between the parental accessions of our mapping populations and among B. nigra reference genomes. Our study is the first one to identify a single locus potentially involved in HR-like cell death induced by insect eggs in B. nigra. Further fine-mapping, comparative genomics and validation of the PEK locus will shed light on the role of these TNL receptors in egg-killing HR.

Genetic analysis reveals three novel QTLs underpinning a butterfly egg-induced hypersensitive response-like cell death in Brassica rapa.

BACKGROUND: Cabbage white butterflies (Pieris spp.) can be severe pests of Brassica crops such as Chinese cabbage, Pak choi (Brassica rapa) or cabbages (B. oleracea). Eggs of Pieris spp. can induce a hypersensitive response-like (HR-like) cell death which reduces egg survival in the wild black mustard (B. nigra). Unravelling the genetic basis of this egg-killing trait in Brassica crops could improve crop resistance to herbivory, reducing major crop losses and pesticides use. Here we investigated the genetic architecture of a HR-like cell death induced by P. brassicae eggs in B. rapa. RESULTS: A germplasm screening of 56 B. rapa accessions, representing the genetic and geographical diversity of a B. rapa core collection, showed phenotypic variation for cell death. An image-based phenotyping protocol was developed to accurately measure size of HR-like cell death and was then used to identify two accessions that consistently showed weak (R-o-18) or strong cell death response (L58). Screening of 160 RILs derived from these two accessions resulted in three novel QTLs for Pieris brassicae-induced cell death on chromosomes A02 (Pbc1), A03 (Pbc2), and A06 (Pbc3). The three QTLs Pbc1-3 contain cell surface receptors, intracellular receptors and other genes involved in plant immunity processes, such as ROS accumulation and cell death formation. Synteny analysis with A. thaliana suggested that Pbc1 and Pbc2 are novel QTLs associated with this trait, while Pbc3 also contains an ortholog of LecRK-I.1, a gene of A. thaliana previously associated with cell death induced by a P. brassicae egg extract. CONCLUSIONS: This study provides the first genomic regions associated with the Pieris egg-induced HR-like cell death in a Brassica crop species. It is a step closer towards unravelling the genetic basis of an egg-killing crop resistance trait, paving the way for breeders to further fine-map and validate candidate genes.

Insect egg-killing: a new front on the evolutionary arms-race between brassicaceous plants and pierid butterflies.

Evolutionary arms-races between plants and insect herbivores have long been proposed to generate key innovations such as plant toxins and detoxification mechanisms that can drive diversification of the interacting species. A novel front-line of plant defence is the killing of herbivorous insect eggs. We test whether an egg-killing plant trait has an evolutionary basis in such a plant-insect arms-race. Within the crucifer family (Brassicaceae), some species express a hypersensitive response (HR)-like necrosis underneath butterfly eggs (Pieridae) that leads to eggs desiccating or falling off the plant. We studied the phylogenetic distribution of this trait, its egg-killing effect on and elicitation by butterflies, by screening 31 Brassicales species, and nine Pieridae species. We show a clade-specific induction of strong, egg-killing HR-like necrosis mainly in species of the Brassiceae tribe including Brassica crops and close relatives. The necrosis is strongly elicited by pierid butterflies that are specialists of crucifers. Furthermore, HR-like necrosis is linked to PR1 defence gene expression, accumulation of reactive oxygen species and cell death, eventually leading to egg-killing. Our findings suggest that the plants' egg-killing trait is a new front on the evolutionary arms-race between Brassicaceae and pierid butterflies beyond the well-studied plant toxins that have evolved against their caterpillars.

Architecture and Dynamics of the Jasmonic Acid Gene Regulatory Network.

Jasmonic acid (JA) is a critical hormonal regulator of plant growth and defense. To advance our understanding of the architecture and dynamic regulation of the JA gene regulatory network, we performed a high-resolution RNA-seq time series of methyl JA-treated Arabidopsis thaliana at 15 time points over a 16-h period. Computational analysis showed that methyl JA (MeJA) induces a burst of transcriptional activity, generating diverse expression patterns over time that partition into distinct sectors of the JA response targeting specific biological processes. The presence of transcription factor (TF) DNA binding motifs correlated with specific TF activity during temporal MeJA-induced transcriptional reprogramming. Insight into the underlying dynamic transcriptional regulation mechanisms was captured in a chronological model of the JA gene regulatory network. Several TFs, including MYB59 and bHLH27, were uncovered as early network components with a role in pathogen and insect resistance. Analysis of subnetworks surrounding the TFs ORA47, RAP2.6L, MYB59, and ANAC055, using transcriptome profiling of overexpressors and mutants, provided insights into their regulatory role in defined modules of the JA network. Collectively, our work illuminates the complexity of the JA gene regulatory network, pinpoints and validates previously unknown regulators, and provides a valuable resource for functional studies on JA signaling components in plant defense and development.

Arabidopsis JASMONATE-INDUCED OXYGENASES down-regulate plant immunity by hydroxylation and inactivation of the hormone jasmonic acid.

The phytohormone jasmonic acid (JA) is vital in plant defense and development. Although biosynthesis of JA and activation of JA-responsive gene expression by the bioactive form JA-isoleucine have been well-studied, knowledge on JA metabolism is incomplete. In particular, the enzyme that hydroxylates JA to 12-OH-JA, an inactive form of JA that accumulates after wounding and pathogen attack, is unknown. Here, we report the identification of four paralogous 2-oxoglutarate/Fe(II)-dependent oxygenases in Arabidopsis thaliana as JA hydroxylases and show that they down-regulate JA-dependent responses. Because they are induced by JA we named them JASMONATE-INDUCED OXYGENASES (JOXs). Concurrent mutation of the four genes in a quadruple Arabidopsis mutant resulted in increased defense gene expression and increased resistance to the necrotrophic fungus Botrytis cinerea and the caterpillar Mamestra brassicae In addition, root and shoot growth of the plants was inhibited. Metabolite analysis of leaves showed that loss of function of the four JOX enzymes resulted in overaccumulation of JA and in reduced turnover of JA into 12-OH-JA. Transformation of the quadruple mutant with each JOX gene strongly reduced JA levels, demonstrating that all four JOXs inactivate JA in plants. The in vitro catalysis of 12-OH-JA from JA by recombinant enzyme could be confirmed for three JOXs. The identification of the enzymes responsible for hydroxylation of JA reveals a missing step in JA metabolism, which is important for the inactivation of the hormone and subsequent down-regulation of JA-dependent defenses.

Genome-wide association study reveals novel players in defense hormone crosstalk in Arabidopsis.

Jasmonic acid (JA) regulates plant defenses against necrotrophic pathogens and insect herbivores. Salicylic acid (SA) and abscisic acid (ABA) can antagonize JA-regulated defenses, thereby modulating pathogen or insect resistance. We performed a genome-wide association (GWA) study on natural genetic variation in Arabidopsis thaliana for the effect of SA and ABA on the JA pathway. We treated 349 Arabidopsis accessions with methyl JA (MeJA), or a combination of MeJA and either SA or ABA, after which expression of the JA-responsive marker gene PLANT DEFENSIN1.2 (PDF1.2) was quantified as a readout for GWA analysis. Both hormones antagonized MeJA-induced PDF1.2 in the majority of the accessions but with a large variation in magnitude. GWA mapping of the SA- and ABA-affected PDF1.2 expression data revealed loci associated with crosstalk. GLYI4 (encoding a glyoxalase) and ARR11 (encoding an Arabidopsis response regulator involved in cytokinin signalling) were confirmed by T-DNA insertion mutant analysis to affect SA-JA crosstalk and resistance against the necrotroph Botrytis cinerea. In addition, At1g16310 (encoding a cation efflux family protein) was confirmed to affect ABA-JA crosstalk and susceptibility to Mamestra brassicae herbivory. Collectively, this GWA study identified novel players in JA hormone crosstalk with potential roles in the regulation of pathogen or insect resistance.

Assessing the Role of ETHYLENE RESPONSE FACTOR Transcriptional Repressors in Salicylic Acid-Mediated Suppression of Jasmonic Acid-Responsive Genes.

Salicylic acid (SA) and jasmonic acid (JA) cross-communicate in the plant immune signaling network to finely regulate induced defenses. In Arabidopsis, SA antagonizes many JA-responsive genes, partly by targeting the ETHYLENE RESPONSE FACTOR (ERF)-type transcriptional activator ORA59. Members of the ERF transcription factor family typically bind to GCC-box motifs in the promoters of JA- and ethylene-responsive genes, thereby positively or negatively regulating their expression. The GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Here, we investigated whether SA-induced ERF-type transcriptional repressors, which may compete with JA-induced ERF-type activators for binding at the GCC-box, play a role in SA/JA antagonism. We selected ERFs that are transcriptionally induced by SA and/or possess an EAR transcriptional repressor motif. Several of the 16 ERFs tested suppressed JA-dependent gene expression, as revealed by enhanced JA-induced PDF1.2 or VSP2 expression levels in the corresponding erf mutants, while others were involved in activation of these genes. However, SA could antagonize JA-induced PDF1.2 or VSP2 in all erf mutants, suggesting that the tested ERF transcriptional repressors are not required for SA/JA cross-talk. Moreover, a mutant in the co-repressor TOPLESS, that showed reduction in repression of JA signaling, still displayed SA-mediated antagonism of PDF1.2 and VSP2. Collectively, these results suggest that SA-regulated ERF transcriptional repressors are not essential for antagonism of JA-responsive gene expression by SA. We further show that de novo SA-induced protein synthesis is required for suppression of JA-induced PDF1.2, pointing to SA-stimulated production of an as yet unknown protein that suppresses JA-induced transcription.

Ethylene: Traffic Controller on Hormonal Crossroads to Defense.

Ethylene (ET) is an important hormone in plant responses to microbial pathogens and herbivorous insects, and in the interaction of plants with beneficial microbes and insects. Early ET signaling events during these biotic interactions involve activities of mitogen-activated protein kinases and ETHYLENE RESPONSE FACTOR transcription factors. Rather than being the principal regulator, ET often modulates defense signaling pathways, including those regulated by jasmonic acid and salicylic acid. Hormonal signal integrations with ET steer the defense signaling network to activate specific defenses that can have direct effects on attackers, or systemically prime distant plant parts for enhanced defense against future attack. ET also regulates volatile signals that attract carnivorous enemies of herbivores or warn neighboring plants. Conversely, ET signaling can also be exploited by attackers to hijack the defense signaling network to suppress effective defenses. In this review, we summarize recent findings on the significant role of ET in the plants' battle against their enemies.

The Snf1-related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress.

The sucrose non-fermenting-1-related protein kinase 2 (SnRK2) family represents a unique family of plant-specific protein kinases implicated in cellular signalling in response to osmotic stress. In our studies, we observed that two class 1 SnRK2 kinases, SnRK2.4 and SnRK2.10, are rapidly and transiently activated in Arabidopsis roots after exposure to salt. Under saline conditions, snrk2.4 knockout mutants had a reduced primary root length, while snrk2.10 mutants exhibited a reduction in the number of lateral roots. The reduced lateral root density was found to be a combinatory effect of a decrease in the number of lateral root primordia and an increase in the number of arrested lateral root primordia. The phenotypes were in agreement with the observed expression patterns of genomic yellow fluorescent protein (YFP) fusions of SnRK2.10 and -2.4, under control of their native promoter sequences. SnRK2.10 was found to be expressed in the vascular tissue at the base of a developing lateral root, whereas SnRK2.4 was expressed throughout the root, with higher expression in the vascular system. Salt stress triggered a rapid re-localization of SnRK2.4-YFP from the cytosol to punctate structures in root epidermal cells. Differential centrifugation experiments of isolated Arabidopsis root proteins confirmed recruitment of endogenous SnRK2.4/2.10 to membranes upon exposure to salt, supporting their observed binding affinity for the phospholipid phosphatidic acid. Together, our results reveal a role for SnRK2.4 and -2.10 in root growth and architecture in saline conditions.

Eggsplorer: a rapid plant-insect resistance determination tool using an automated whitefly egg quantification algorithm.

BACKGROUND: A well-known method for evaluating plant resistance to insects is by measuring insect reproduction or oviposition. Whiteflies are vectors of economically important viral diseases and are, therefore, widely studied. In a common experiment, whiteflies are placed on plants using clip-on-cages, where they can lay hundreds of eggs on susceptible plants in a few days. When quantifying whitefly eggs, most researchers perform manual eye measurements using a stereomicroscope. Compared to other insect eggs, whitefly eggs are many and very tiny, usually 0.2 mm in length and 0.08 mm in width; therefore, this process takes a lot of time and effort with and without prior expert knowledge. Plant insect resistance experiments require multiple replicates from different plant accessions; therefore, an automated and rapid method for quantifying insect eggs can save time and human resources. RESULTS: In this work, a novel automated tool for fast quantification of whitefly eggs is presented to accelerate the determination of plant insect resistance and susceptibility. Leaf images with whitefly eggs were collected from a commercial microscope and a custom-built imaging system. A deep learning-based object detection model was trained using the collected images. The model was incorporated into an automated whitefly egg quantification algorithm, deployed in a web-based application called Eggsplorer. Upon evaluation on a testing dataset, the algorithm was able to achieve a counting accuracy as high as 0.94, r2 of 0.99, and a counting error of ± 3 eggs relative to the actual number of eggs counted by eye. The automatically collected counting results were used to determine the resistance and susceptibility of several plant accessions and were found to yield significantly comparable results as when using the manually collected counts for analysis. CONCLUSION: This is the first work that presents a comprehensive step-by-step method for fast determination of plant insect resistance and susceptibility with the assistance of an automated quantification tool.

Tetraose steroidal glycoalkaloids from potato provide resistance against Alternaria solani and Colorado potato beetle.

Plants with innate disease and pest resistance can contribute to more sustainable agriculture. Natural defence compounds produced by plants have the potential to provide a general protective effect against pathogens and pests, but they are not a primary target in resistance breeding. Here, we identified a wild relative of potato, Solanum commersonii, that provides us with unique insight in the role of glycoalkaloids in plant immunity. We cloned two atypical resistance genes that provide resistance to Alternaria solani and Colorado potato beetle through the production of tetraose steroidal glycoalkaloids (SGA). Moreover, we provide in vitro evidence to show that these compounds have potential against a range of different (potato pathogenic) fungi. This research links structural variation in SGAs to resistance against potato diseases and pests. Further research on the biosynthesis of plant defence compounds in different tissues, their toxicity, and the mechanisms for detoxification, can aid the effective use of such compounds to improve sustainability of our food production.

Farmers often rely on pesticides to protect their crops from disease and pests. However, these chemicals are harmful to the environment and more sustainable strategies are needed. This is particularly true for a disease known as the early blight of potato, which is primarily treated using fungicides that stop the fungal pathogen responsible for the infection (Alternaria solani) from growing. An alternative approach is to harness the natural defence systems that plants already have in place to protect themselves. Like humans, plants have an immune system which can detect and destroy specific pathogens. On top of this, they release defence compounds that are generally toxic to pests and microbes, stopping them from infiltrating and causing an infection. In 2021, a group of researchers discovered a wild relative of the potato, known as Solanum commersonii, with strong resistance to early blight disease. Here, Wolters et al. ­ including some of the researchers involved in the 2021 study ­ set out to find how this plant defends itself from the fungus A. solani. The team found that two closely linked genes are responsible for the resistant behaviour of S. commersonii, which both encode enzymes known as glycosyltransferases. Further experiments revealed that the enzymes protect S. commersonii from early blight disease by modifying steroidal glycoalkaloids, typical defence compounds found in potato and other plants from the same family. The glycosyltransferases alter glycoalkaloids in S. commersonii by adding a sugar group to a specific part of the compound called glycone. Wolters et al. found that the glycoalkaloids from S. commersonii were able to slow the growth of other fungal pathogens that harm potatoes when tested in the laboratory. They also made plants resistant to another common destroyer of crops, the Colorado potato beetle. These findings could help farmers breed potatoes and other crops that are more resistant to early blight disease and Colorado potato beetle, as well as potentially other fungi and pests. However, further experiments are needed to investigate how these glycone-modified glycoalkaloids affect humans, and how variants of glycoalkaloids are produced and degraded in different parts of the plants. Acquiring this knowledge will help to employ these defence compounds in a safe and effective manner.

How salicylic acid takes transcriptional control over jasmonic acid signaling.

Transcriptional regulation is a central process in plant immunity. The induction or repression of defense genes is orchestrated by signaling networks that are directed by plant hormones of which salicylic acid (SA) and jasmonic acid (JA) are the major players. Extensive cross-communication between the hormone signaling pathways allows for fine tuning of transcriptional programs, determining resistance to invaders and trade-offs with plant development. Here, we give an overview of how SA can control transcriptional reprogramming of JA-induced genes in Arabidopsis thaliana. SA can influence activity and/or localization of transcriptional regulators by post-translational modifications of transcription factors and co-regulators. SA-induced redox changes, mediated by thioredoxins and glutaredoxins, modify transcriptional regulators that are involved in suppression of JA-dependent genes, such as NPR1 and TGA transcription factors, which affects their localization or DNA binding activity. Furthermore, SA can mediate sequestering of JA-responsive transcription factors away from their target genes by stalling them in the cytosol or in complexes with repressor proteins in the nucleus. SA also affects JA-induced transcription by inducing degradation of transcription factors with an activating role in JA signaling, as was shown for the ERF transcription factor ORA59. Additionally, SA can induce negative regulators, among which WRKY transcription factors, that can directly or indirectly inhibit JA-responsive gene expression. Finally, at the DNA level, modification of histones by SA-dependent factors can result in repression of JA-responsive genes. These diverse and complex regulatory mechanisms affect important signaling hubs in the integration of hormone signaling networks. Some pathogens have evolved effectors that highjack hormone crosstalk mechanisms for their own good, which are described in this review as well.