Regulation of Glandular Size and Phytoalexin Biosynthesis by a Negative Feedback Loop in Cotton.

Plants have evolved diverse defense mechanisms encompassing physical and chemical barriers. Cotton pigment glands are known for containing various defense metabolites, but the precise regulation of gland size to modulate defense compound levels remains enigmatic. Here, it is discovered that the VQ domain-containing protein JAVL negatively regulates pigment gland size and the biosynthesis of defense compounds, while the MYC2-like transcription factor GoPGF has the opposite effect. Notably, GoPGF directly activates the expression of JAVL, whereas JAVL suppresses GoPGF transcription, establishing a negative feedback loop that maintains the expression homeostasis between GoPGF and JAVL. Furthermore, it is observed that JAVL negatively regulates jasmonate levels by inhibiting the expression of jasmonate biosynthetic genes and interacting with GoPGF to attenuate its activation effects, thereby maintaining homeostatic regulation of jasmonate levels. The increased expression ratio of GoPGF to JAVL leads to enlarged pigment glands and elevated jasmonates and defense compounds, enhancing insect and pathogen resistance in cotton. These findings unveil a new mechanism for regulating gland size and secondary metabolites biosynthesis, providing innovative strategies for strengthening plant defense.

The proteomic landscape of fall armyworm oral secretion reveals its role in plant adaptation.

BACKGROUND: The fall armyworm (FAW, Spodoptera frugiperda (J.E. Smith)) is a polyphagous agricultural pest with rapidly evolving adaptations to host plants. We found the oral secretion (OS) of FAW from different plants influences plant defense response differentially, suggesting its role in adapting to host plants. However, the protein expression profile of FAW OS respond to different plants is largely unknown. RESULTS: Here, from the mass spectrometry assay, we identified a total of 256 proteins in the OS of FAW fed on cotton (Gossypium hirsutum L.), tobacco (Nicotiana benthamiana Domin), maize (Zea mays L.) and artificial diet. The FAW OS primarily comprise of 60 proteases, 32 esterases and 92 non-enzymatic proteins. It displays high plasticity across different diets. We found that more than half of the esterases are lipases which have been reported as insect elicitors to enhance plant defense response. The lipase accumulation in cotton-fed larvae was the highest, followed by maize-fed larvae. In the presence of lipase inhibitors, the enhanced induction on defense genes in wounded leaves by OS was attenuated. However, the putative effectors were most highly accumulated in the OS from FAW larvae fed on maize compared to those fed on other diets. We identified that one of them (VRLP4) reduces the OS-mediated induction on defense genes in wounded leaves. CONCLUSION: Together, our investigation presents the proteomic landscape of the OS of FAW influenced by different diets and reveals diet-mediated plasticity of OS is involved in FAW adaptation to host plants. © 2024 Society of Chemical Industry.

Endocytosis-mediated entry of a caterpillar effector into plants is countered by Jasmonate.

Insects and pathogens release effectors into plant cells to weaken the host defense or immune response. While the imports of some bacterial and fungal effectors into plants have been previously characterized, the mechanisms of how caterpillar effectors enter plant cells remain a mystery. Using live cell imaging and real-time protein tracking, we show that HARP1, an effector from the oral secretions of cotton bollworm (Helicoverpa armigera), enters plant cells via protein-mediated endocytosis. The entry of HARP1 into a plant cell depends on its interaction with vesicle trafficking components including CTL1, PATL2, and TET8. The plant defense hormone jasmonate (JA) restricts HARP1 import by inhibiting endocytosis and HARP1 loading into endosomes. Combined with the previous report that HARP1 inhibits JA signaling output in host plants, it unveils that the effector and JA establish a defense and counter-defense loop reflecting the robust arms race between plants and insects.

A highly accumulated secretory protein from cotton bollworm interacts with basic helix-loop-helix transcription factors to dampen plant defense.

Caterpillar oral secretion (OS) contains active molecules that modulate plant defense signaling. We isolated an effector-like protein (Highly Accumulated Secretory Protein 1, HAS1) from cotton bollworm (Helicoverpa armigera) that is the most highly accumulated secretory protein of the nondigestive components in OS and belongs to venom R-like protein. Elimination of HAS1 by plant-mediated RNA interference reduced the suppression of OS on the defense response in plants. Plants expressing HAS1 are more susceptible to insect herbivory accompanied by the reduced expressions of multiple defense genes. HAS1 binds to the basic helix-loop-helix (bHLH) transcription factors, including GoPGF involved in pigmented gland formation and defense compounds biosynthesis in cotton and MYC3/MYC4 the main regulators in jasmonate (JA) signaling in Arabidopsis. The binding activity is required for HAS1 to inhibit the activation of bHLHs on plant defense gene expressions. Together with our previous study that another venom R-like protein HARP1 in cotton bollworm OS blocks JA signaling by interacting with JASMONATE-ZIM-domain repressors, we conclude that the venom R-like proteins in OS interfere with plant defense in a dual suppression manner. Considering the venom proteins in parasitic wasp assault the immune system of its host animal, our investigation reveals their conserved function in carnivorous and herbivorous insects.

Differential Contributions of MYCs to Insect Defense Reveals Flavonoids Alleviating Growth Inhibition Caused by Wounding in Arabidopsis.

In Arabidopsis, basic helix-loop-helix transcription factors (TFs) MYC2, MYC3, and MYC4 are involved in many biological processes, such as defense against insects. We found that despite functional redundancy, MYC-related mutants displayed different resistance to cotton bollworm (Helicoverpa armigera). To screen out the most likely genes involved in defense against insects, we analyzed the correlation of gene expression with cotton bollworm resistance in wild-type (WT) and MYC-related mutants. In total, the expression of 94 genes in untreated plants and 545 genes in wounded plants were strongly correlated with insect resistance, and these genes were defined as MGAIs (MYC-related genes against insects). MYC3 had the greatest impact on the total expression of MGAIs. Gene ontology (GO) analysis revealed that besides the biosynthesis pathway of glucosinolates (GLSs), MGAIs, which are well-known defense compounds, were also enriched in flavonoid biosynthesis. Moreover, MYC3 dominantly affected the gene expression of flavonoid biosynthesis. Weighted gene co-expression network analysis (WGCNA) revealed that AAE18, which is involved in activating auxin precursor 2,4-dichlorophenoxybutyric acid (2,4-DB) and two other auxin response genes, was highly co-expressed with flavonoid biosynthesis genes. With wounding treatment, the WT plants exhibited better growth performance than chalcone synthase (CHS), which was defective in flavonoid biosynthesis. The data demonstrated dominant contributions of MYC3 to cotton bollworm resistance and imply that flavonoids might alleviate the growth inhibition caused by wounding in Arabidopsis.

Differential Transcription and Alternative Splicing in Cotton Underly Specialized Defense Responses Against Pests.

The green mirid bug (Apolygus lucorum) and the cotton bollworm (Helicoverpa armigera) are both preferred to live on cotton but cause different symptoms, suggesting specialized responses of cotton to the two insects. In this study, we investigated differential molecular mechanisms underlying cotton plant defenses against A. lucorum and H. armigera via transcriptomic analyses. At the transcription level, jasmonate (JA) signaling was dominated in defense against H. armigera whereas salicylic acid (SA) signaling was more significant in defense against A. lucorum. A set of pathogenesis-related (PR) genes and protease inhibitor genes were differentially induced by the two insects. Insect infestations also had an impact on alternative splicing (AS), which was altered more significantly by the H. armigera than A. lucorum. Interestingly, most differential AS (DAS) genes had no obvious change at the transcription level. GO analysis revealed that biological process termed "RNA splicing" and "cellular response to abiotic stimulus" were enriched only in DAS genes from the H. armigera infested samples. Furthermore, insect infestations induced the retained intron of GhJAZs transcripts, which produced a truncated protein lacking the intact Jas motif. Taken together, our data demonstrate that the specialized cotton response to different insects is regulated by gene transcription and AS as well.

Research advances in plant-insect molecular interaction.

Acute and precise signal perception and transduction are essential for plant defense against insects. Insect elicitors-that is, the biologically active molecules from insects' oral secretion (which contains regurgitant and saliva), frass, ovipositional fluids, and the endosymbionts-are recognized by plants and subsequently induce a local or systematic defense response. On the other hand, insects secrete various types of effectors to interfere with plant defense at multiple levels for better adaptation. Jasmonate is a main regulator involved in plant defense against insects and integrates with multiple pathways to make up the intricate defense network. Jasmonate signaling is strictly regulated in plants to avoid the hypersensitive defense response and seems to be vulnerable to assault by insect effectors at the same time. Here, we summarize recently identified elicitors, effectors, and their target proteins in plants and discuss their underlying molecular mechanisms.

AP2/ERF Transcription Factors Integrate Age and Wound Signals for Root Regeneration.

Age and wounding are two major determinants for regeneration. In plants, the root regeneration is triggered by wound-induced auxin biosynthesis. As plants age, the root regenerative capacity gradually decreases. How wounding leads to the auxin burst and how age and wound signals collaboratively regulate root regenerative capacity are poorly understood. Here, we show that the increased levels of three closely-related miR156-targeted Arabidopsis (Arabidopsis thaliana) SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, SPL2, SPL10, and SPL11, suppress root regeneration with age by inhibiting wound-induced auxin biosynthesis. Mechanistically, we find that a subset of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors including ABSCISIC ACID REPRESSOR1 and ERF109 is rapidly induced by wounding and serves as a proxy for wound signal to induce auxin biosynthesis. In older plants, SPL2/10/11 directly bind to the promoters of AP2/ERFs and attenuates their induction, thereby dampening auxin accumulation at the wound. Our results thus identify AP2/ERFs as a hub for integration of age and wound signal for root regeneration.

Plant Specialized Metabolism Regulated by Jasmonate Signaling.

As sessile and autotrophic organisms, plants have evolved sophisticated pathways to produce a rich array of specialized metabolites, many of which are biologically active and function as defense substances in protecting plants from herbivores and pathogens. Upon stimuli, these structurally diverse small molecules may be synthesized or constitutively accumulated. Jasmonate acids (JAs) are the major defense phytohormone involved in transducing external signals (such as wounding) to activate defense reactions, including, in particular, the reprogramming of metabolic pathways that initiate and enhance the production of defense compounds against insect herbivores and pathogens. In this review, we summarize the progress of recent research on the control of specialized metabolic pathways in plants by JA signaling, with an emphasis on the molecular regulation of terpene and alkaloid biosynthesis. We also discuss the interplay between JA signaling and various signaling pathways during plant defense responses. These studies provide valuable data for breeding insect-proof crops and pave the way to engineering the production of valuable metabolites in future.

An effector from cotton bollworm oral secretion impairs host plant defense signaling.

Insects have evolved effectors to conquer plant defense. Most known insect effectors are isolated from sucking insects, and examples from chewing insects are limited. Moreover, the targets of insect effectors in host plants remain unknown. Here, we address a chewing insect effector and its working mechanism. Cotton bollworm (Helicoverpa armigera) is a lepidopteran insect widely existing in nature and severely affecting crop productivity. We isolated an effector named HARP1 from H. armigera oral secretion (OS). HARP1 was released from larvae to plant leaves during feeding and entered into the plant cells through wounding sites. Expression of HARP1 in Arabidopsis mitigated the global expression of wounding and jasmonate (JA) responsive genes and rendered the plants more susceptible to insect feeding. HARP1 directly interacted with JASMONATE-ZIM-domain (JAZ) repressors to prevent the COI1-mediated JAZ degradation, thus blocking JA signaling transduction. HARP1-like proteins have conserved function as effectors in noctuidae, and these types of effectors might contribute to insect adaptation to host plants during coevolution.

A gossypol biosynthetic intermediate disturbs plant defence response.

Plant secondary metabolites and their biosynthesis have attracted great interest, but investigations of the activities of hidden intermediates remain rare. Gossypol and related sesquiterpenes are the major phytoalexins in cotton. Among the six biosynthetic intermediates recently identified, 8-hydroxy-7-keto-δ-cadinene (C234) crippled the plant disease resistance when accumulated upon gene silencing. C234 harbours an α,ß-unsaturated carbonyl thus is a reactive electrophile species. Here, we show that C234 application also dampened the Arabidopsis resistance against the bacterial pathogen Pseudomonas syringae pv. maculicola ( Psm). We treated Arabidopsis with C234, Psm and ( Psm+C234), and analysed the leaf transcriptomes. While C234 alone exerted a mild effect, it greatly stimulated an over-response to the pathogen. Of the 7335 genes affected in the ( Psm+C234)-treated leaves, 3476 were unresponsive without the chemical, in which such functional categories as 'nucleotides transport', 'vesicle transport', 'MAP kinases', 'G-proteins', 'protein assembly and cofactor ligation' and 'light reaction' were enriched, suggesting that C234 disturbed certain physiological processes and the protein complex assembly, leading to distorted defence response and decreased disease resistance. As C234 is efficiently metabolized by CYP71BE79, plants of cotton lineage have evolved a highly active enzyme to prevent the phytotoxic intermediate accumulation during gossypol pathway evolution. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

Characterization of gossypol biosynthetic pathway.

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion.

Core cis-element variation confers subgenome-biased expression of a transcription factor that functions in cotton fiber elongation.

Cotton cultivars have evolved to produce extensive, long, seed-born fibers important for the textile industry, but we know little about the molecular mechanism underlying spinnable fiber formation. Here, we report how PACLOBUTRAZOL RESISTANCE 1 (PRE1) in cotton, which encodes a basic helix-loop-helix (bHLH) transcription factor, is a target gene of spinnable fiber evolution. Differential expression of homoeologous genes in polyploids is thought to be important to plant adaptation and novel phenotypes. PRE1 expression is specific to cotton fiber cells, upregulated during their rapid elongation stage and A-homoeologous biased in allotetraploid cultivars. Transgenic studies demonstrated that PRE1 is a positive regulator of fiber elongation. We determined that the natural variation of the canonical TATA-box, a regulatory element commonly found in many eukaryotic core promoters, is necessary for subgenome-biased PRE1 expression, representing a mechanism underlying the selection of homoeologous genes. Thus, variations in the promoter of the cell elongation regulator gene PRE1 have contributed to spinnable fiber formation in cotton. Overexpression of GhPRE1 in transgenic cotton yields longer fibers with improved quality parameters, indicating that this bHLH gene is useful for improving cotton fiber quality.

Arabidopsis Transcription Factors SPL1 and SPL12 Confer Plant Thermotolerance at Reproductive Stage.

Plant reproductive organs are vulnerable to heat, but regulation of heat-shock responses in inflorescence is largely uncharacterized. Here, we report that two of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcriptional factors in Arabidopsis, SPL1 and SPL12, act redundantly in thermotolerance at the reproductive stage. The spl1-1 spl12-1 inflorescences displayed hypersensitivity to heat stress, whereas overexpression of SPL1 or SPL12 enhanced the thermotolerance in both Arabidopsis and tobacco. RNA sequencing revealed 1939 upregulated and 1479 downregulated genes in wild-type inflorescence upon heat stress, among which one-quarter (1,040) was misregulated in spl1-1 spl12-1, indicating that SPL1 and SPL12 contribute greatly to the heat-triggered transcriptional reprogramming in inflorescence. Notably, heat stress induced a large number of abscisic acid (ABA) responsive genes, of which ∼39% were disturbed in heat induction in spl1-1 spl12-1 inflorescence. Preapplication of ABA and overexpression of SPL1 restored the inflorescence thermotolerance in spl1-1 spl12-1 and in the ABA biosynthesis mutant aba2-1, but not in the pyl sextuple mutant defective in ABA receptors PYR1/PYL1/PYL2/PYL4/PYL5/PYL8. Thus, inflorescence thermotolerance conferred by SPL1 and SPL2 involves PYL-mediated ABA signaling. The molecular network consisting of SPL1 and SPL12 illustrated here shed new light on the mechanisms of plant thermotolerance at the reproductive stage.

Jasmonate response decay and defense metabolite accumulation contributes to age-regulated dynamics of plant insect resistance.

Immunity deteriorates with age in animals but comparatively little is known about the temporal regulation of plant resistance to herbivores. The phytohormone jasmonate (JA) is a key regulator of plant insect defense. Here, we show that the JA response decays progressively in Arabidopsis. We show that this decay is regulated by the miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9) group of proteins, which can interact with JA ZIM-domain (JAZ) proteins, including JAZ3. As SPL9 levels gradually increase, JAZ3 accumulates and the JA response is attenuated. We provide evidence that this pathway contributes to insect resistance in young plants. Interestingly however, despite the decay in JA response, older plants are still comparatively more resistant to both the lepidopteran generalist Helicoverpa armigera and the specialist Plutella xylostella, along with increased accumulation of glucosinolates. We propose a model whereby constitutive accumulation of defense compounds plays a role in compensating for age-related JA-response attenuation during plant maturation.

Targeting insect mitochondrial complex I for plant protection.

Plant engineered to express double-stranded RNA (dsRNA) can target the herbivorous insect gene for silencing. Although mounting evidence has emerged to support feasibility of this new pest control technology, field application is slow largely due to lack of potent targets. Here, we show that suppression of the gene encoding NDUFV2, a subunit of mitochondrial complex I that catalyses NADH dehydrogenation in respiratory chain, was highly lethal to insects. Feeding cotton bollworm (Helicoverpa armigera) larvae with transgenic cotton tissues expressing NDUFV2 dsRNA led to mortality up to 80% within 5 days, and almost no larvae survived after 7 days of feeding, due to the altered mitochondrial structure and activity. Transcriptome comparisons showed a drastic repression of dopa decarboxylase genes. Reciprocal assays with Asian corn borer (Ostrinia furnacalis), another lepidopteran species, revealed the sequence-specific effect of NDUFV2 suppression. Furthermore, the hemipteran lugus Apolygus lucorum was also liable to NDUFV2 repression. These data demonstrate that the mitochondrial complex I is a promising target with both sequence specificity and wide applicability for the development of new-generation insect-proof crops.

Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites.

Of the two cultivated species of allopolyploid cotton, Gossypium barbadense produces extra-long fibers for the production of superior textiles. We sequenced its genome (AD)2 and performed a comparative analysis. We identified three bursts of retrotransposons from 20 million years ago (Mya) and a genome-wide uneven pseudogenization peak at 11-20 Mya, which likely contributed to genomic divergences. Among the 2,483 genes preferentially expressed in fiber, a cell elongation regulator, PRE1, is strikingly At biased and fiber specific, echoing the A-genome origin of spinnable fiber. The expansion of the PRE members implies a genetic factor that underlies fiber elongation. Mature cotton fiber consists of nearly pure cellulose. G. barbadense and G. hirsutum contain 29 and 30 cellulose synthase (CesA) genes, respectively; whereas most of these genes (>25) are expressed in fiber, genes for secondary cell wall biosynthesis exhibited a delayed and higher degree of up-regulation in G. barbadense compared with G. hirsutum, conferring an extended elongation stage and highly active secondary wall deposition during extra-long fiber development. The rapid diversification of sesquiterpene synthase genes in the gossypol pathway exemplifies the chemical diversity of lineage-specific secondary metabolites. The G. barbadense genome advances our understanding of allopolyploidy, which will help improve cotton fiber quality.

Interaction between two timing microRNAs controls trichome distribution in Arabidopsis.

The miR156-targeted squamosa promoter binding protein like (SPL) transcription factors function as an endogenous age cue in regulating plant phase transition and phase-dependent morphogenesis, but the control of SPL output remains poorly understood. In Arabidopsis thaliana the spatial pattern of trichome is a hallmark of phase transition and governed by SPLs. Here, by dissecting the regulatory network controlling trichome formation on stem, we show that the miR171-targeted lost meristems 1 (LOM1), LOM2 and LOM3, encoding GRAS family members previously known to maintain meristem cell polarity, are involved in regulating the SPL activity. Reduced LOM abundance by overexpression of miR171 led to decreased trichome density on stems and floral organs, and conversely, constitutive expression of the miR171-resistant LOM (rLOM) genes promoted trichome production, indicating that LOMs enhance trichome initiation at reproductive stage. Genetic analysis demonstrated LOMs shaping trichome distribution is dependent on SPLs, which positively regulate trichome repressor genes TRICHOMELESS 1 (TCL1) and TRIPTYCHON (TRY). Physical interaction between the N-terminus of LOMs and SPLs underpins the repression of SPL activity. Importantly, other growth and developmental events, such as flowering, are also modulated by LOM-SPL interaction, indicating a broad effect of the LOM-SPL interplay. Furthermore, we provide evidence that MIR171 gene expression is regulated by its targeted LOMs, forming a homeostatic feedback loop. Our data uncover an antagonistic interplay between the two timing miRNAs in controlling plant growth, phase transition and morphogenesis through direct interaction of their targets.