Adaptation of Helicoverpa armigera to Soybean Peptidase Inhibitors Is Associated with the Transgenerational Upregulation of Serine Peptidases.

Molecular phenotypes induced by environmental stimuli can be transmitted to offspring through epigenetic inheritance. Using transcriptome profiling, we show that the adaptation of Helicoverpa armigera larvae to soybean peptidase inhibitors (SPIs) is associated with large-scale gene expression changes including the upregulation of genes encoding serine peptidases in the digestive system. Furthermore, approximately 60% of the gene expression changes induced by SPIs persisted in the next generation of larvae fed on SPI-free diets including genes encoding regulatory, oxidoreductase, and protease functions. To investigate the role of epigenetic mechanisms in regulating SPI adaptation, the methylome of the digestive system of first-generation larvae (fed on a diet with and without SPIs) and of the progeny of larvae exposed to SPIs were characterized. A comparative analysis between RNA-seq and Methyl-seq data did not show a direct relationship between differentially methylated and differentially expressed genes, while trypsin and chymotrypsin genes were unmethylated in all treatments. Rather, DNA methylation potential epialleles were associated with transcriptional and translational controls; these may play a regulatory role in the adaptation of H. armigera to SPIs. Altogether, our findings provided insight into the mechanisms of insect adaptation to plant antiherbivore defense proteins and illustrated how large-scale transcriptional reprograming of insect genes can be transmitted across generations.

Sugarcane multitrophic interactions: Integrating belowground and aboveground organisms.

Sugarcane is a crop of major importance used mainly for sugar and biofuel production, and many additional applications of its byproducts are being developed. Sugarcane cultivation is plagued by many insect pests and pathogens that reduce sugarcane yields overall. Recently emerging studies have shown complex multitrophic interactions in cultivated areas, such as the induction of sugarcane defense-related proteins by insect herbivory that function against fungal pathogens that commonly appear after mechanical damage. Fungi and viruses infecting sugarcane also modulate insect behavior, for example, by causing changes in volatile compounds responsible for insect attraction or repelling natural vector enemies via a mechanism that increases pathogen dissemination from infected plants to healthy ones. Interestingly, the fungus Fusarium verticillioides is capable of being vertically transmitted to insect offspring, ensuring its persistence in the field. Understanding multitrophic complexes is important to develop better strategies for controlling pathosystems affecting sugarcane and other important crops and highlights the importance of not only studying binary interactions but also adding as many variables as possible to effectively translate laboratory research to real-life conditions.

BAK1 is involved in AtRALF1-induced inhibition of root cell expansion.

The rapid alkalinization factor (RALF) peptide negatively regulates cell expansion, and an antagonistic relationship has been demonstrated between AtRALF1, a root-specific RALF isoform in Arabidopsis, and brassinosteroids (BRs). An evaluation of the response of BR signaling mutants to AtRALF1 revealed that BRI1-associated receptor kinase1 (bak1) mutants are insensitive to AtRALF1 root growth inhibition activity. BAK1 was essential for the induction of AtRALF1-responsive genes but showed no effect on the mobilization of Ca2+ and alkalinization responses. Homozygous plants accumulating AtRALF1 and lacking the BAK1 gene did not exhibit the characteristic semi-dwarf phenotype of AtRALF1-overexpressors. Biochemical evidence indicates that AtRALF1 and BAK1 physically interact with a Kd of 4.6 µM and acridinium-labeled AtRALF1 was used to demonstrate that part of the specific binding of AtRALF1 to intact seedlings and to a microsomal fraction derived from the roots of Arabidopsis plants is BAK1-dependent. Moreover, AtRALF1 induces an increase in BAK1 phosphorylation, suggesting that the binding of AtRALF1 to BAK1 is functional. These findings show that BAK1 contains an additional AtRALF1 binding site, indicating that this protein may be part of a AtRALF1-containing complex as a co-receptor, and it is required for the negative regulation of cell expansion.

Arabidopsis thaliana rapid alkalinization factor 1-mediated root growth inhibition is dependent on calmodulin-like protein 38.

Arabidopsis thaliana rapid alkalinization factor 1 (AtRALF1) is a small secreted peptide hormone that inhibits root growth by repressing cell expansion. Although it is known that AtRALF1 binds the plasma membrane receptor FERONIA and conveys its signals via phosphorylation, the AtRALF1 signaling pathway is largely unknown. Here, using a yeast two-hybrid system to search for AtRALF1-interacting proteins in Arabidopsis, we identified calmodulin-like protein 38 (CML38) as an AtRALF1-interacting partner. We also found that CML38 and AtRALF1 are both secreted proteins that physically interact in a Ca2+- and pH-dependent manner. CML38-knockout mutants generated via T-DNA insertion were insensitive to AtRALF1, and simultaneous treatment with both AtRALF1 and CML38 proteins restored sensitivity in these mutants. Hybrid plants lacking CML38 and having high accumulation of the AtRALF1 peptide did not exhibit the characteristic short-root phenotype caused by AtRALF1 overexpression. Although CML38 was essential for AtRALF1-mediated root inhibition, it appeared not to have an effect on the AtRALF1-induced alkalinization response. Moreover, acridinium-labeling of AtRALF1 indicated that the binding of AtRALF1 to intact roots is CML38-dependent. In summary, we describe a new component of the AtRALF1 response pathway. The new component is a calmodulin-like protein that binds AtRALF1, is essential for root growth inhibition, and has no role in AtRALF1 alkalinization.

Defense-related proteins involved in sugarcane responses to biotic stress.

Sugarcane is one of the most important agricultural crops in the world. However, pathogen infection and herbivore attack cause constant losses in yield. Plants respond to pathogen infection by inducing the expression of several protein types, such as glucanases, chitinases, thaumatins, peptidase inhibitors, defensins, catalases and glycoproteins. Proteins induced by pathogenesis are directly or indirectly involved in plant defense, leading to pathogen death or inducing other plant defense responses. Several of these proteins are induced in sugarcane by different pathogens or insects and have antifungal or insecticidal activity. In this review, defense-related proteins in sugarcane are described, with their putative mechanisms of action, pathogen targets and biotechnological perspectives.

Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding.

Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory.

Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation.

Rapid alkalinization factor (RALF) is a peptide signal that plays a basic role in cell biology and most likely regulates cell expansion. In this study, transgenic Arabidopsis thaliana lines with high and low levels of AtRALF1 transcripts were used to investigate this peptide's mechanism of action. Overexpression of the root-specific isoform AtRALF1 resulted in reduced cell size. Conversely, AtRALF1 silencing increased root length by increasing the size of root cells. AtRALF1-silenced plants also showed an increase in the number of lateral roots, whereas AtRALF1 overexpression produced the opposite effect. In addition, four AtRALF1-inducible genes were identified: two genes encoding proline-rich proteins (AtPRP1 and AtPRP3), one encoding a hydroxyproline-rich glycoprotein (AtHRPG2), and one encoding a xyloglucan endotransglucosylase (TCH4). These genes were expressed in roots and involved in cell-wall rearrangement, and their induction was concentration dependent. Furthermore, AtRALF1-overexpressing plants were less sensitive to exogenous brassinolide (BL); upon BL treatment, the plants showed no increase in root length and a compromised increase in hypocotyl elongation. In addition, the treatment had no effect on the number of emerged lateral roots. AtRALF1 also induces two brassinosteroid (BR)-downregulated genes involved in the BR biosynthetic pathway: the cytochrome P450 monooxygenases CONSTITUTIVE PHOTOMORPHISM AND DWARFISM (CPD) and DWARF4 (DWF4). Simultaneous treatment with both AtRALF1 and BL caused a reduction in AtRALF1-inducible gene expression levels, suggesting that these signals may compete for components shared by both pathways. Taken together, these results indicate an opposing effect of AtRALF1 and BL, and suggest that RALF's mechanism of action could be to interfere with the BR signalling pathway.

The Phytopathogen Fusarium verticillioides Modifies the Intestinal Morphology of the Sugarcane Borer.

BACKGROUND: In tropical sugarcane crops, the fungus Fusarium verticillioides, the agent responsible for the occurrence of the red rot complex, occurs in association with the sugarcane borer Diatraea saccharalis. This fungus, in addition to being transmitted vertically, can manipulate both the insect and the plant for its own dissemination in the field. Due to the complex interaction between F. verticillioides and D. saccharalis, and the high incidence of the fungus in the intestinal region, our objective was to investigate whether F. verticillioides could alter the intestinal structure of the insect. METHODS: We combined analysis of scanning electron microscopy and light microscopy to identify whether the presence of the fungus F. verticillioides, in artificial diets or in sugarcane, could lead to any alteration or regional preference in the insect's intestinal ultrastructure over the course of its development, or its offspring development, analyzing the wall and microvillous structures of the mid-digestive system. RESULTS: Here, we show that the fungus F. verticillioides alters the intestinal morphology of D. saccharalis, promoting an increase of up to 3.3 times in the thickness of the midgut compared to the control. We also observed that the phytopathogen colonizes the intestinal microvilli for reproduction, suggesting that this region can be considered the gateway of the fungus to the insect's reproductive organs. In addition, the colonization of this region promoted the elongation of microvillous structures by up to 180% compared to the control, leading to an increase in the area used for colonization. We also used the fungus Colletotrichum falcatum in the tests, and it did not differ from the control in any test, showing that this interaction is specific between D. saccharalis and F. verticillioides. CONCLUSIONS: The phytopathogenic host F. verticillioides alters the intestinal morphology of the vector insect in favor of its colonization.

FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex.

The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl-truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.

Sugarwin: a sugarcane insect-induced gene with antipathogenic activity.

In sugarcane fields, colonization of the stalk by opportunistic fungi usually occurs after the caterpillar Diatraea saccharalis attacks the sugarcane plant. Plants respond to insect attack by inducing and accumulating a large set of defense proteins. Two homologues of a barley wound-inducible protein (BARWIN), sugarcane wound-inducible proteins SUGARWIN1 and SUGARWIN2, have been identified in sugarcane by an in silico analysis. Antifungal properties have been described for a number of BARWIN homologues. We report that a SUGARWIN::green fluorescent protein fusion protein is located in the endoplasmic reticulum and in the extracellular space of sugarcane plants. The induction of sugarwin transcripts occurs in response to mechanical wounding, D. saccharalis damage, and methyl jasmonate treatment. The accumulation of transcripts is late induced and is restricted to the site of the wound. Although the transcripts of sugarwin genes were strongly increased following insect attack, the protein itself did not show any effect on insect development; rather, it altered fungal morphology, leading to the apoptosis of the germlings. These results suggest that, in the course of evolution, sugarwin-encoding genes were recruited by sugarcane due to their antipathogenic activity. We rationalize that sugarcane is able to induce sugarwin gene expression in response to D. saccharalis feeding as a concerted plant response to the anticipated invasion by the fungi that typically penetrate the plant stalk after insect damage.

Evolutionary history of Arabidopsis thaliana aminoacyl-tRNA synthetase dual-targeted proteins.

Aminoacyl-transfer RNA (tRNA) synthetases (aaRS) are key players in translation and act early in protein synthesis by mediating the attachment of amino acids to their cognate tRNA molecules. In plants, protein synthesis may occur in three subcellular compartments (cytosol, mitochondria, and chloroplasts), which requires multiple versions of the protein to be correctly delivered to its proper destination. The organellar aaRS are nuclear encoded and equipped with targeting information at the N-terminal sequence, which enables them to be specifically translocated to their final location. Most of the aaRS families present organellar proteins that are dual targeted to mitochondria and chloroplasts. Here, we examine the dual targeting behavior of aaRS from an evolutionary perspective. Our results show that Arabidopsis thaliana aaRS sequences are a result of a horizontal gene transfer event from bacteria. However, there is no evident bias indicating one single ancestor (Cyanobacteria or Proteobacteria). The dual-targeted aaRS phylogenetic relationship was characterized into two different categories (paralogs and homologs) depending on the state recovered for both dual-targeted and cytosolic proteins. Taken together, our results suggest that the dual-targeted condition is a gain-of-function derived from gene duplication. Selection may have maintained the original function in at least one of the copies as the additional copies diverged.

Colletotrichum falcatum modulates the olfactory behavior of the sugarcane borer, favoring pathogen infection.

Some pathogens can manipulate their host plants and insects to optimize their fitness, increasing the attraction of insects to the infected plant in ways that facilitate pathogen acquisition. In tropical American sugarcane crops, the fungus Colletotrichum falcatum, the red rot causal agent, usually occurs in association with the sugarcane borer Diatraea saccharalis, resulting in large losses of this crop. Considering this association, we aimed to identify the effects of C. falcatum on D. saccharalis host preference and performance as well as the effect of this insect on C. falcatum sugarcane infection. Here, we show that the fungus C. falcatum modulates D. saccharalis behavior to its own benefit. More specifically, C. falcatum-infected sugarcane plants showed a dramatic increase in VOCs, luring D. saccharalis females to lay eggs on these plants. Therefore, sugarcane infection by the fungus C. falcatum increased in cooccurrence with insect herbivory, benefiting the pathogen when associated with D. saccharalis.

Root secretion of defense-related proteins is development-dependent and correlated with flowering time.

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.

Fungal phytopathogen modulates plant and insect responses to promote its dissemination.

Vector-borne plant pathogens often change host traits to manipulate vector behavior in a way that favors their spread. By contrast, infection by opportunistic fungi does not depend on vectors, although damage caused by an herbivore may facilitate infection. Manipulation of hosts and vectors, such as insect herbivores, has not been demonstrated in interactions with fungal pathogens. Herein, we establish a new paradigm for the plant-insect-fungus association in sugarcane. It has long been assumed that Fusarium verticillioides is an opportunistic fungus, where it takes advantage of the openings left by Diatraea saccharalis caterpillar attack to infect the plant. In this work, we show that volatile emissions from F. verticillioides attract D. saccharalis caterpillars. Once they become adults, the fungus is transmitted vertically to their offspring, which continues the cycle by inoculating the fungus into healthy plants. Females not carrying the fungus prefer to lay their eggs on fungus-infected plants than mock plants, while females carrying the fungus prefer to lay their eggs on mock plants than fungus-infected plants. Even though the fungus impacts D. saccharalis sex behavior, larval weight and reproduction rate, most individuals complete their development. Our data demonstrate that the fungus manipulates both the host plant and insect herbivore across life cycle to promote its infection and dissemination.

Structural and Evolutionary Analyses of PR-4 SUGARWINs Points to a Different Pattern of Protein Function.

SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by Diatraea saccharalis larvae. These proteins play an important role in plant defense, in particular against fungal pathogens, such as Colletothricum falcatum (Went) and Fusarium verticillioides. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) [H44 and H146, SUGARWINs] in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.

SacRALF1, a peptide signal from the grass sugarcane (Saccharum spp.), is potentially involved in the regulation of tissue expansion.

Rapid alkalinization factor (RALF) is part of a growing family of small peptides with hormone characteristics in plants. Initially isolated from leaves of tobacco plants, RALF peptides can be found throughout the plant kingdom and they are expressed ubiquitously in plants. We took advantage of the small gene family size of RALF genes in sugarcane and the ordered cellular growth of the grass sugarcane leaves to gain information about the function of RALF peptides in plants. Here we report the isolation of two RALF peptides from leaves of sugarcane plants using the alkalinization assay. SacRALF1 was the most abundant and, when added to culture media, inhibited growth of microcalli derived from cell suspension cultures at concentrations as low as 0.1 microM. Microcalli exposed to exogenous SacRALF1 for 5 days showed a reduced number of elongated cells. Only four copies of SacRALF genes were found in sugarcane plants. All four SacRALF genes are highly expressed in young and expanding leaves and show a low or undetectable level of expression in expanded leaves. In half-emerged leaf blades, SacRALF transcripts were found at high levels at the basal portion of the leaf and at low levels at the apical portion. Gene expression analyzes localize SacRALF genes in elongation zones of roots and leaves. Mature leaves, which are devoid of expanding cells, do not show considerable expression of SacRALF genes. Our findings are consistent with SacRALF genes playing a role in plant development potentially regulating tissue expansion.

AtPIN: Arabidopsis thaliana protein interaction network.

BACKGROUND: Protein-protein interactions (PPIs) constitute one of the most crucial conditions to sustain life in living organisms. To study PPI in Arabidopsis thaliana we have developed AtPIN, a database and web interface for searching and building interaction networks based on publicly available protein-protein interaction datasets. DESCRIPTION: All interactions were divided into experimentally demonstrated or predicted. The PPIs in the AtPIN database present a cellular compartment classification (C3) which divides the PPI into 4 classes according to its interaction evidence and subcellular localization. It has been shown in the literature that a pair of genuine interacting proteins are generally expected to have a common cellular role and proteins that have common interaction partners have a high chance of sharing a common function. In AtPIN, due to its integrative profile, the reliability index for a reported PPI can be postulated in terms of the proportion of interaction partners that two proteins have in common. For this, we implement the Functional Similarity Weight (FSW) calculation for all first level interactions present in AtPIN database. In order to identify target proteins of cytosolic glutamyl-tRNA synthetase (Cyt-gluRS) (AT5G26710) we combined two approaches, AtPIN search and yeast two-hybrid screening. Interestingly, the proteins glutamine synthetase (AT5G35630), a disease resistance protein (AT3G50950) and a zinc finger protein (AT5G24930), which has been predicted as target proteins for Cyt-gluRS by AtPIN, were also detected in the experimental screening. CONCLUSIONS: AtPIN is a friendly and easy-to-use tool that aggregates information on Arabidopsis thaliana PPIs, ontology, and sub-cellular localization, and might be a useful and reliable strategy to map protein-protein interactions in Arabidopsis. AtPIN can be accessed at http://bioinfo.esalq.usp.br/atpin.

Conservation of dual-targeted proteins in Arabidopsis and rice points to a similar pattern of gene-family evolution.

Gene duplication followed by acquisition of specific targeting information and dual targeting were evolutionary strategies enabling organelles to cope with overlapping functions. We examined the evolutionary trend of dual-targeted single-gene products in Arabidopsis and rice genomes. The number of paralogous proteins encoded by gene families and the dual-targeted orthologous proteins were analysed. The number of dual-targeted proteins and the corresponding gene-family sizes were similar in Arabidopsis and rice irrespective of genome sizes. We show that dual targeting of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase was maintained despite occurrence of whole-genome duplications in Arabidopsis and rice as well as a polyploidization followed by a diploidization event (gene loss) in the latter.

A conserved dibasic site is essential for correct processing of the peptide hormone AtRALF1 in Arabidopsis thaliana.

Prohormone proteins in animals and yeast are typically processed at dibasic sites by convertases. Propeptide hormones are also found in plants but little is known about processing. We show for the first time that a dibasic site upstream of a plant peptide hormone, AtRALF1, is essential for processing. Overexpression of preproAtRALF1 causes semi-dwarfism whereas overexpression of preproAtRALF1(R69A), the propeptide with a mutation in the dibasic site, shows a normal phenotype. RALF1(R69A) plants accumulate only the mutated proprotein and not the processed peptide. In vitro processing using microsomal fractions suggests that processing is carried out by a kexin-like convertase.