Host adaptation and specialization in Tetranychidae mites.

Composite generalist herbivores are comprised of host-adapted populations that retain the ability to shift hosts. The degree and overlap of mechanisms used by host-adapted generalist and specialist herbivores to overcome the same host plant defenses are largely unknown. Tetranychidae mites are exceptionally suited to address the relationship between host adaptation and specialization in herbivores as this group harbors closely related species with remarkably different host ranges-an extreme generalist the two-spotted spider mite (Tetranychus urticae Koch [Tu]) and the Solanaceous specialist Tetranychus evansi (Te). Here, we used tomato-adapted two-spotted spider mite (Tu-A) and Te populations to compare mechanisms underlying their host adaptation and specialization. We show that both mites attenuate induced tomato defenses, including protease inhibitors (PIs) that target mite cathepsin L digestive proteases. While Te solely relies on transcriptional attenuation of PI induction, Tu and Tu-A have elevated constitutive activity of cathepsin L proteases, making them less susceptible to plant anti-digestive proteins. Tu-A and Te also rely on detoxification of tomato constitutive defenses. Te uses esterase and P450 activities, while Tu-A depends on the activity of all major detoxification enzymatic classes to disarm tomato defensive compounds to a lesser extent. Thus, even though both Tu-A and Te use similar mechanisms to counteract tomato defenses, Te can better cope with them. This finding is congruent with the ecological and evolutionary times required to establish mite adaptation and specialization states, respectively.

Jasmonic Acid-Dependent MYC Transcription Factors Bind to a Tandem G-Box Motif in the YUCCA8 and YUCCA9 Promoters to Regulate Biotic Stress Responses.

The indole-3-pyruvic acid pathway is the main route for auxin biosynthesis in higher plants. Tryptophan aminotransferases (TAA1/TAR) and members of the YUCCA family of flavin-containing monooxygenases catalyze the conversion of l-tryptophan via indole-3-pyruvic acid to indole-3-acetic acid (IAA). It has been described that jasmonic acid (JA) locally produced in response to mechanical wounding triggers the de novo formation of IAA through the induction of two YUCCA genes, YUC8 and YUC9. Here, we report the direct involvement of a small number of basic helix-loop-helix transcription factors of the MYC family in this process. We show that the JA-mediated regulation of the expression of the YUC8 and YUC9 genes depends on the abundance of MYC2, MYC3, and MYC4. In support of this observation, seedlings of myc knockout mutants displayed a strongly reduced response to JA-mediated IAA formation. Furthermore, transactivation assays provided experimental evidence for the binding of MYC transcription factors to a particular tandem G-box motif abundant in the promoter regions of YUC8 and YUC9, but not in the promoters of the other YUCCA isogenes. Moreover, we demonstrate that plants that constitutively overexpress YUC8 and YUC9 show less damage after spider mite infestation, thereby underlining the role of auxin in plant responses to biotic stress signals.

Silencing barley cystatins HvCPI-2 and HvCPI-4 specifically modifies leaf responses to drought stress.

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy-2 and Icy-4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy-2 and Icy-4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap-1, HvPap-12, and HvPap-16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.

Phytocystatins: Defense Proteins against Phytophagous Insects and Acari.

This review deals with phytocystatins, focussing on their potential role as defence proteins against phytophagous arthropods. Information about the evolutionary, molecular and biochemical features and inhibitory properties of phytocystatins are presented. Cystatin ability to inhibit heterologous cysteine protease activities is commented on as well as some approaches of tailoring cystatin specificity to enhance their defence function towards pests. A general landscape on the digestive proteases of phytophagous insects and acari and the remarkable plasticity of their digestive physiology after feeding on cystatins are highlighted. Biotechnological approaches to produce recombinant cystatins to be added to artificial diets or to be sprayed as insecticide-acaricide compounds and the of use cystatins as transgenes are discussed. Multiple examples and applications are included to end with some conclusions and future perspectives.

Plant protein peptidase inhibitors: an evolutionary overview based on comparative genomics.

BACKGROUND: Peptidases are key proteins involved in essential plant physiological processes. Although protein peptidase inhibitors are essential molecules that modulate peptidase activity, their global presence in different plant species remains still unknown. Comparative genomic analyses are powerful tools to get advanced knowledge into the presence and evolution of both, peptidases and their inhibitors across the Viridiplantae kingdom. RESULTS: A genomic comparative analysis of peptidase inhibitors and several groups of peptidases in representative species of different plant taxonomic groups has been performed. The results point out: i) clade-specific presence is common to many families of peptidase inhibitors, being some families present in most land plants; ii) variability is a widespread feature for peptidase inhibitory families, with abundant species-specific (or clade-specific) gene family proliferations; iii) peptidases are more conserved in different plant clades, being C1A papain and S8 subtilisin families present in all species analyzed; and iv) a moderate correlation among peptidases and their inhibitors suggests that inhibitors proliferated to control both endogenous and exogenous peptidases. CONCLUSIONS: Comparative genomics has provided valuable insights on plant peptidase inhibitor families and could explain the evolutionary reasons that lead to the current variable repertoire of peptidase inhibitors in specific plant clades.

Cysteine peptidases and their inhibitors in Tetranychus urticae: a comparative genomic approach.

BACKGROUND: Cysteine peptidases in the two-spotted spider mite Tetranychus urticae are involved in essential physiological processes, including proteolytic digestion. Cystatins and thyropins are inhibitors of cysteine peptidases that modulate their activity, although their function in this species has yet to be investigated. Comparative genomic analyses are powerful tools to obtain advanced knowledge into the presence and evolution of both, peptidases and their inhibitors, and could aid to elucidate issues concerning the function of these proteins. RESULTS: We have performed a genomic comparative analysis of cysteine peptidases and their inhibitors in T. urticae and representative species of different arthropod taxonomic groups. The results indicate: i) clade-specific proliferations are common to C1A papain-like peptidases and for the I25B cystatin family of inhibitors, whereas the C1A inhibitors thyropins are evolutionarily more conserved among arthropod clades; ii) an unprecedented extensive expansion for C13 legumain-like peptidases is found in T. urticae; iii) a sequence-structure analysis of the spider mite cystatins suggests that diversification may be related to an expansion of their inhibitory range; and iv) an in silico transcriptomic analysis shows that most cathepsin B and L cysteine peptidases, legumains and several members of the cystatin family are expressed at a higher rate in T. urticae feeding stages than in embryos. CONCLUSION: Comparative genomics has provided valuable insights on the spider mite cysteine peptidases and their inhibitors. Mite-specific proliferations of C1A and C13 peptidase and I25 cystatin families and their over-expression in feeding stages of mites fit with a putative role in mite's feeding and could have a key role in its broad host feeding range.

Transcriptome analysis of chestnut (Castanea sativa) tree buds suggests a putative role for epigenetic control of bud dormancy.

BACKGROUND AND AIMS: Recent papers indicated that epigenetic control is involved in transitions in bud dormancy, purportedly controlling gene expression. The present study aimed to identify genes that are differentially expressed in dormant and non-dormant Castanea sativa buds. METHODS: Two suppression subtractive hybridization cDNA libraries were constructed to characterize the transcriptomes of dormant apical buds of C. sativa, and buds in which dormancy was released. KEY RESULTS: A total of 512 expressed sequence tags (ESTs) were generated in a forward and reverse subtractive hybridization experiment. Classification of these ESTs into functional groups demonstrated that dormant buds were predominantly characterized by genes associated with stress response, while non-dormant buds were characterized by genes associated with energy, protein synthesis and cellular components for development and growth. ESTs for a few genes involved in different forms of epigenetic modification were found in both libraries, suggesting a role for epigenetic control in bud dormancy different from that in growth. Genes encoding histone mono-ubiquitinase HUB2 and histone acetyltransferase GCN5L were associated with dormancy, while a gene encoding histone H3 kinase AUR3 was associated with growth. Real-time RT-PCR with a selection of genes involved in epigenetic modification and stress tolerance confirmed the expression of the majority of investigated genes in various stages of bud development, revealing a cyclical expression pattern concurring with the growth seasons for most genes. However, senescing leaves also showed an increased expression of several of the genes associated with dormancy, implying pleiotropy. Furthermore, a comparison between these subtraction cDNA libraries and the poplar bud dormancy transcriptome and arabidopsis transcriptomes for seed dormancy and non-dormancy indicated a common basis for dormancy in all three systems. CONCLUSIONS: Bud dormancy and non-dormancy in C. sativa were characterized by distinct sets of genes and are likely to be under different epigenetic control.

Gene pyramiding of peptidase inhibitors enhances plant resistance to the spider mite Tetranychus urticae.

The two-spotted spider mite Tetranychus urticae is a damaging pest worldwide with a wide range of host plants and an extreme record of pesticide resistance. Recently, the complete T. urticae genome has been published and showed a proliferation of gene families associated with digestion and detoxification of plant secondary compounds which supports its polyphagous behaviour. To overcome spider mite adaptability a gene pyramiding approach has been developed by co-expressing two barley proteases inhibitors, the cystatin Icy6 and the trypsin inhibitor Itr1 genes in Arabidopsis plants by Agrobacterium-mediated transformation. The presence and expression of both transgenes was studied by conventional and quantitative real time RT-PCR assays and by indirect ELISA assays. The inhibitory activity of cystatin and trypsin inhibitor was in vitro analysed using specific substrates. Single and double transformants were used to assess the effects of spider mite infestation. Double transformed lines showed the lowest damaged leaf area in comparison to single transformants and non-transformed controls and different accumulation of H(2)O(2) as defence response in the leaf feeding site, detected by diaminobenzidine staining. Additionally, an impact on endogenous mite cathepsin B- and L-like activities was observed after feeding on Arabidopsis lines, which correlates with a significant increase in the mortality of mites fed on transformed plants. These effects were analysed in view of the expression levels of the target mite protease genes, C1A cysteine peptidase and S1 serine peptidase, identified in the four developmental mite stages (embryo, larvae, nymphs and adults) performed using the RNA-seq information available at the BOGAS T. urticae database. The potential of pyramiding different classes of plant protease inhibitors to prevent plant damage caused by mites as a new tool to prevent pest resistance and to improve pest control is discussed.