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Molecular phenotypes induced by environmental stimuli can be transmitted to offspring through epigenetic inheritance. Using transcriptome profiling, we show that the adaptation of

Insects are known plant pests, and some of them such as Trichoplusia ni feed on a variety of crops. In this study, Trichoplusia ni was fed distinct diets of leaves of Arabidopsis thaliana or Solanum lycopersicum as well as an artificial diet. After four generations, the microbial composition of the insect gut was evaluated to determine if the diet influenced the structure and function of the microbial communities. The population fed with A. thaliana had higher proportions of Shinella, Terribacillus and Propionibacterium, and these genera are known to have tolerance to glucosinolate activity, which is produced by A. thaliana to deter insects. The population fed with S. lycopersicum expressed increased relative abundances of the Agrobacterium and Rhizobium genera. These microbial members can degrade alkaloids, which are produced by S. lycopersicum. All five of these genera were also present in the respective leaves of either A. thaliana or S. lycopersicum, suggesting that these microbes are acquired by the insects from the diet itself. This study describes a potential mechanism used by generalist insects to become habituated to their available diet based on acquisition of phytochemical degrading gut bacteria.

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.

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.

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.

Serine peptidases (SPs) are responsible for most primary protein digestion in Lepidoptera species. An expansion of the number of genes encoding trypsin and chymotrypsin enzymes and the ability to upregulate the expression of some of these genes in response to peptidase inhibitor (PI) ingestion have been associated with the adaptation of Noctuidae moths to herbivory. To investigate whether these gene family expansion events are common to other Lepidoptera groups, we searched for all genes encoding putative trypsin and chymotrypsin enzymes in 23 publicly available genomes from this taxon. Phylogenetic analysis showed that several gene family expansion events may have occurred in the taxon&rsquo, s evolutionary history and that these events gave rise to a very diverse group of enzymes, including proteins lacking the canonical SP catalytic triad. The expression profile of these enzymes along the midgut and the secretion mechanisms by which these enzymes enter the luminal content were also analyzed in Spodoptera frugiperda larvae using RNA-seq and proteomics. These results support the proposal of a midgut countercurrent flux responsible for the direction of these proteins to the anterior portion of the midgut and show that these enzymes reach the midgut lumen via both exocytosis and microapocrine secretion mechanisms.

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.

SUGARWIN1 and 2 are defense proteins from sugarcane. Their gene expression is known to be induced in response to wound and Diatraea saccharalis damage. Although the recombinant SUGARWIN protein does not affect insect development, it promotes significant morphological and physiological changes in Fusarium verticillioides and Colletotrichum falcatum, which lead to fungal cell death via apoptosis. In this study, we deepen our understanding of the role of SUGARWINs in plant defense and the molecular mechanisms by which these proteins affect fungi by elucidating their molecular targets. Our results show that SUGARWINs play an important role in plant defense against opportunistic pathogens. We demonstrated that SUGARWINs are induced by C. falcatum, and the induction of SUGARWINs can vary among sugarcane varieties. The sugarcane variety exhibiting the highest level of SUGARWIN induction exhibited a considerable reduction in C. falcatum infection. Furthermore, SUGARWIN1 exhibited ribonuclease, chitosanase, and chitinase activity, whereas SUGARWIN2 exhibited only chitosanase activity. This variable enzymatic specificity seems to be the result of divergent amino acid composition within the substrate-binding site.

Peptidase inhibitors (PIs) are essential proteins involved in plant resistance to herbivorous insects, yet many insect species are able to escape the negative effects of these molecules. We compared the effects of acute and chronic ingestion of soybean peptidase inhibitors (SPIs) on Spodoptera frugiperda and Diatraea saccharalis, two Lepidoptera species with different sensitivities to SPI ingestion. We analyzed the trypsin and chymotrypsin gene expression profiles in both species. Acute exposure of S. frugiperda to the inhibitors activated seven genes (SfChy5, SfChy9, SfChy19, SfChy22, SfTry6, SfTry8, and SfTry10), whereas chronic exposure activated 16 genes (SfChy2, SfChy4, SfChy5, SfChy8, SfChy9, SfChy11, SfChy12, SfChy15, SfChy17, SfChy21, SfChy22, SfTry6, SfTry8, SfTry9, SfTry10, and SfTry12). By contrast, the challenge of D. saccharalis with SPIs did not differentially induce the expression of trypsin- or chymotrypsin-encoding genes, with the exception of DsChy7. Bayesian phylogenetic analysis of S. frugiperda trypsin protein sequences revealed two gene clades: one composed of genes responsive to the SPIs and a second composed of the unresponsive genes. D. saccharalis trypsin proteins were clustered nearest to the S. frugiperda unresponsive genes. Overall, our findings support a hypothesized mechanism of resistance of Noctuidae moths to SPIs, involving gene number expansion of trypsin and chymotrypsin families and regulation of gene expression, which could also explain the variable susceptibility between S. frugiperda and D. saccharalis to these plant inhibitors.

Many plant genes have their expression modulated by stress conditions. Here, we used Arabidopsis FtsH5 protease, which expression is regulated by light stress, as bait in a yeast two-hybrid screen to search for new proteins involved in the stress response. As a result, we found FIP (FtsH5 Interacting Protein), which possesses an amino proximal cleavable transit peptide, a hydrophobic membrane-anchoring region, and a carboxyl proximal C4-type zinc-finger domain. In vivo experiments using FIP fused to green fluorescent protein (GFP) showed a plastid localization. This finding was corroborated by chloroplast import assays that showed FIP inserted in the thylakoid membrane. FIP expression was down-regulated in plants exposed to high light intensity, oxidative, salt, and osmotic stresses, whereas mutant plants expressing low levels of FIP were more tolerant to these abiotic stresses. Our data shows a new thylakoid-membrane protein involved with abiotic stress response in Arabidopsis thaliana.

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic L-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

The overwhelming demand for food requires the application of technology on field. An important issue that limits the productivity of crops is related to insect attacks. Hence, several studies have evaluated the application of different compounds to reduce the field losses, especially insecticide compounds from plant sources. Among them, plant protease inhibitors (PIs) have been studied in both basic and applied researches, displaying positive results in control of some insects. However, certain species are able to bypass the insecticide effects exerted by PIs. In this review, we disclosed the adaptive mechanisms showed by lepidopteran and coleopteran insects, the most expressive insect orders related to crop predation. The structural aspects involved in adaptation mechanisms are presented as well as the newest alternatives for pest control. The application of biotechnological tools in crop protection will be mandatory in agriculture, and it will be up to researchers to find the best candidates for effective control in long-term.

Many plant genes have their expression modulated by stress conditions. Here, we used Arabidopsis FtsH5 protease, which expression is regulated by light stress, as bait in a yeast two-hybrid screen to search for new proteins involved in the stress response. As a result, we found FIP (FtsH5 Interacting Protein), which possesses an amino proximal cleavable transit peptide, a hydrophobic membrane-anchoring region, and a carboxyl proximal C4-type zinc-finger domain. In vivo experiments using FIP fused to green fluorescent protein (GFP) showed a plastid localization. This finding was corroborated by chloroplast import assays that showed FIP inserted in the thylakoid membrane. FIP expression was down-regulated in plants exposed to high light intensity, oxidative, salt, and osmotic stresses, whereas mutant plants expressing low levels of FIP were more tolerant to these abiotic stresses. Our data shows a new thylakoid-membrane protein involved with abiotic stress response in Arabidopsis thaliana.

Under environmental conditions, plants are constantly exposed to a wide range of biotic interactions, which include insects, and pathogens. Usually scientists are tempted to study each association individually, which reduces the complexity of the interaction. This restricted view of the problem does not consider that plants are the ballroom in which a multitude of organisms are constantly interacting with each other affecting not only plant responses but also how one organism responds to the other. Plants attacked by insects and pathogens display profound physiological, morphological and chemical changes or adaptations that result in organism attraction or avoidance, depending on the species involved. Therefore, many researchers worldwide have decided to study this phenomenon in a more holistic view, integrating genetics, ecology and physiology to depict these complex interactions. In this review, we will discuss how plant infection by pathogens may affect insect behavior and vice-versa and how plants cope with these multitude of biotic stresses.

Summary The peptide RALF regulates cell expansion via an as-yet-unknown mechanism. Here, we provide evidence that AtRALF1 may be negatively regulating cell expansion by interfering with the brassinosteroid signalling pathway., 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.

0The fall armyworm (Spodoptera frugiperda) is an important pest insect due to high degree of polyphagia. In order to better understand its adaptation mechanism against plant protease inhibitors, bioassays were carried supplementing diet with the Kunitz trypsin inhibitor from Entada acaciifolia seeds (EATI). In vitro assays showed an increase of proteolytic activity in EATI-fed larvae midgut. Moreover, the trypsin enzymes showed insensitivity to inhibition with EATI. In order to understand what genes were overexpressed after chronic exposition to EATI, quantitative RT-PCR analyses were performed and revealed an increase in transcription of two trypsin genes, suggesting its participation in insensitivity of midgut trypsins. Another important result was the expression of one chymotrypsin gene, which is not expressed in control fed-larvae but induced in EATI-fed larvae. New regions of higher molecular weight showing proteolytic activity were visualized in inhibitor-fed larvae by zymography gel electrophoresis, proposing that the new enzymes expressed in response of inhibitor dietary would be formatting oligomers. This is a characteristic also observed in other pest insects that adapt to feed in plant protease inhibitors diet. Additional assays revealed that trypsins from EATI-fed larvae also became insensitive against Kunitz and Bowman-Birk inhibitors from soybean. This result suggests a possible involvement of the same S. frugiperda genes in adaptation against Kunitz and Bowman-Birk inhibitors in their host plants.

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.

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.

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.

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.

Sugarcane is a proven biofuel feedstock and accounts for about 40% of the biofuel production worldwide. It has a more favorable energy input/output ratio than that of corn, the other major biofuel feedstock. The rich resource of genetic diversity and the plasticity of autopolyploid genomes offer a wealth of opportunities for the application of genomics and technologies to address fundamental questions in sugarcane towards maximizing biomass production. In a workshop on sugarcane engineering held at Rutgers University, we identified research areas and emerging technologies that could have significant impact on sugarcane improvement. Traditional plant physiological studies and standardized phenotypic characterization of sugarcane are essential for dissecting the developmental processes and patterns of gene expression in this complex polyploid species. Breeder friendly DNA markers associated with target traits will enhance selection efficiency and shorten the long breeding cycles. Integration of cold tolerance from Saccharum spontaneum and Miscanthus has the potential to expand the geographical range of sugarcane production from tropical and subtropical regions to temperate zones. The Flex-stock and mix-stock concepts could be solutions for sustaining local biorefineries where no single biofuel feedstock could provide consistent year-round supplies. The ever increasing capacities of genomics and biotechnologies pave the way for fully exploring these potentials to optimize sugarcane for biofuel production.

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.

The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a "shotgun" approach, based on a general up regulation of a large set of endoproteinases.

A previously described DNA sequence generator algorithm (DNA-SGA) using error-correcting codes has been employed as a computational tool to address the evolutionary pathway of the genetic code. The code-generated sequence alignment demonstrated that a residue mutation revealed by the code can be found in the same position in sequences of distantly related taxa. Furthermore, the code-generated sequences do not promote amino acid changes in the deviant genomes through codon reassignment. A Bayesian evolutionary analysis of both code-generated and homologous sequences of the Arabidopsis thaliana malate dehydrogenase gene indicates an approximately 1 MYA divergence time from the MDH code-generated sequence node to its paralogous sequences. The DNA-SGA helps to determine the plesiomorphic state of DNA sequences because a single nucleotide alteration often occurs in distantly related taxa and can be found in the alternative codon patterns of noncanonical genetic codes. As a consequence, the algorithm may reveal an earlier stage of the evolution of the standard code.

The molecular variability among 10 populations of Spodoptera frugiperda (J.E. Smith), collected from maize, Zea mays L., or cotton Gossypium hirsutum L. crops located at distinctive geographical regions in Brazil, was assessed through random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR). In total, 208 RAPD markers were evaluated, and 98% of them were polymorphic. The mean genetic similarity was 0.6621 and 0.2499 by the Simple Matching and Jaccard matrices, respectively. In general, the unweighted pair-group method with arithmetic average dendrograms separated the populations into clusters related to the geographical origin of the samples. No branch of the dendrograms underpinning a molecular association of S. frugiperda has been identified to either of the two host plants. The molecular variance analysis showed that 18 and 82% of the genetic variation was distributed among and within the groups of populations, respectively. The principal coordinate analysis reinforced the pattern of population clustering found with the unweighted pair-group method with arithmetic average method. These results suggest the occurrence of considerable gene flow between S. frugiperda populations from maize and cotton fields located in the same region in Brazil. Therefore, for an effective management of this pest, there is an urgent need for a better understanding of the gene flow of S. frugiperda populations associated to different host plants along the distribution range of this pest over time in a specific cropping system.

Rapid alkalinization factor (RALF) is a peptide signal that plays a role in plant cell expansion. We have recently proposed that AtRALF1 negatively regulates root cell elongation and lateral root formation by opposing the effects of brassinosteroid (BR). We reported 6 AtRALF1-inducible cell wall-related genes and 2 P450 monooxygenase -encoding genes involved in the BR biosynthetic pathway. The AtRALF1-inducible genes implicated in cell wall remodeling were not downregulated by brassinolide (BL) treatment alone; their induction was only compromised following simultaneous treatment with AtRALF1 and BL. We further examined the cell wall-remodeling gene EXPANSIN A5 (AtEXPA5), which is upregulated by BL and has been shown to positively affect root cell elongation. Herein, we report that AtEXPA5 expression is downregulated by AtRALF1 in a dose-dependent manner in the roots and hypocotyls of Arabidopsis plants. AtEXPA5 is also downregulated in plants that overexpress AtRALF1, and it is upregulated in plants in which the AtRALF1 gene is partially silenced. The AtRALF1 peptide is also able to repress AtEXPA5 induction following a pre-treatment with BL. A schematic diagram showing the gene regulatory network connecting the recently reported genes with the regulation of cell expansion by AtEXPA5 is presented.

The Arabidopsis thaliana THI1 protein is involved in thiamine biosynthesis and is targeted to both chloroplasts and mitochondria by N-terminal control regions. To investigate thi1 expression, a series of thi1 promoter deletions were fused to the beta-glucuronidase (GUS) reporter gene. Transgenic plants were generated and expression patterns obtained under different environmental conditions. The results show that expression derived from the thi1 promoter is detected early on during development and continues throughout the plant's life cycle. High levels of GUS expression are observed in both shoots and roots during vegetative growth although, in roots, expression is restricted to the vascular system. Deletion analysis of the thi1 promoter region identified a region that is responsive to light. The smallest fragment (designated Pthi322) encompasses 306 bp and possesses all the essential signals for tissue specificity, as well as responsiveness to stress conditions such as sugar deprivation, high salinity, and hypoxia.

One of the earliest events in the evolution of mitochondria was the development a means to translocate proteins made in the cytosol into the ‘‘protomitochondrion.’’ How this was achieved remains uncertain, and the nature of the earliest version of the protein translocation machinery is not known. Comparative sequence analysis suggests three subunits, Tom40, Tom7, and Tom22 as common elements of the protein translocase in the mitochondrial outer membrane in diverse extant eukaryotes. Tom22, the 22-kDa subunit, plays a critical role in the function of this complex in fungi and animals, and we show that an 8-kDa subunit of the plant translocase is a truncated form of Tom22. It has a single transmembrane segment conforming in sequence to the same region of Tom22 from other eukaryotic lineages and a short carboxy-terminal trans domain located in the mitochondrial intermembrane space. The trans domain from the Arabidopsis thaliana protein functions in yeast lacking their own Tom22 by complementing protein import defects and restoring cell growth. Moreover, we have identified orthologs of Tom22, Tom7, and Tom40 in diverse eukaryotes such as the diatom Phaeodactylum tricornutum, the amoebic slime Dictyostelium discoideum, and the protozoan parasite Plasmodium falciparum. This finding strongly suggests these subunits as the core of the protein translocase in the earliest mitochondria.

We used bioassays to investigate the effect of Bowman-Birk and Kunitz-type soybean proteinase inhibitors on two artificial diets (diets 1 and 2) which are commonly used to feed laboratory colonies of larvae of the moth Diatraea saccharalis, monitoring food intake and utilization, and larval development and mortality. Diet 1 was less nutritious, with a low protein content and reduced mineral and essential amino acid (e.g., cysteine, lysine, and methionine) content, while diet 2 was richer and more complete. When proteinase inhibitors were incorporated into the artificial diets, the effects on larval development were significantly greater for those larvae fed diet 1, with the chronic ingestion of proteinase inhibitors reducing the level of trypsin-like activity in the midgut of larvae fed this diet. Larvae fed diet 2 also showed a reduced level of tryptic activity in the midgut, but this was less marked than for diet 1. These results indicate that despite their inhibitory effect on midgut enzymes, the effectiveness of proteinase inhibitors is directly dependent on the quality of the diet. The different effects seen on insect biology when proteinase inhibitors are added to rich or poor diets suggests that the role of anti-nutritional proteins in the control of insects might not be adequately addressed by bioassays based on the incorporation of inhibitors into artificial diets.

Cysteine proteinases from larvae of the common bean weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae), were isolated by ion exchange affinity chromatography on a CM-Cellulose column and used to select mutant cystatins from a library made with the filamentous M13 phage display system. The library contained variant cystatins derived from the nematode Onchocerca volvulus cystatin through mutagenesis of loop 1, which contains the QVVAG motif that is involved in binding to proteinases. After three rounds of selection, the activity of variant cystatins against papain and cysteine proteinases from A. obtectus was assayed by ELISA. Two different variant cystatins (presenting amino acids DVVSA and NTSSA at positions 65–69) bound to A. obtectus cysteine proteinases more tightly than to papain. In contrast, the wild type had similar affinity for A. obtectus proteinases and for papain. These two selected variants cystatins have greater specificity towards A. obtectus cysteine proteinases than the original sequence and could represent good candidate genes for the production of transgenic plants resistant to this insect pest.

Previously, we genetically modified two Brazilian commercial sugarcane (Saccharum sp.) varieties (SP80-3280 and SP80-1842) for resistance to the sugarcane borer Diatraea saccharalis by co-bombardment transformation with three plasmids: pCIB4421 containing the Bacillus thuringiensis gene cry1Ab regulated by a maize phosphoenol-pyruvate carboxylase promoter, pCIB4426 carrying cry1Ab controlled by a pith promoter, and pHA9 with the neo antibiotic resistance gene. The genetically modified plants were submitted to molecular characterization and insect feeding bioassays. Plants also were screened in the field for insect infestation and had their phenotypic characteristics and quantitative traits evaluated. This study presents evidence that the B. thuringiensis cry1Ab gene can be efficiently expressed in sugarcane plants over an extended growing period, and can confer resistance to the sugarcane borer under field conditions, while not measurably altering numerous material traits of the crop.

Arabidopsis thaliana THI1 is encoded by a single nuclear gene and directed simultaneously to mitochondria and chloroplasts from a single major transcript. In vitro transcription/translation experiments revealed the presence of two translational products by the differential usage of two in-frame translational start codons. The coupling site-specific mutations on the THI1 encoding sequence with green fluorescent protein (GFP) gene fusions showed that translation initiation at the first AUG directs translocation of THI1 to chloroplasts. However, when translation starts from the second AUG, THI1 is addressed to mitochondria. Analysis of the translation efficiency of thi1 mRNA revealed that the best context for translation initiation is to use the first AUG. In addition, a suboptimal context in the vicinity of the second AUG initiation codon, next to a stable stem-and-loop structure that is likely to slow translation, has been noted. The fact that translation preferentially occurs in the first AUG of this protein suggests a high requirement for TH1 in chloroplasts. Although the frequency of upstream AUG translation is higher, according to the first AUG rule, initiation at the second AUG deviates significantly from Kozak's consensus. It suggests leaky ribosomal scanning, reinitiation or the internal entry of ribosomes to assure mitochondrial protein import.

In this review, plant-insect interaction is discussed as a dynamic system, subjected to continual variation and change. Plants developed different mechanisms to reduce insect attack, including specific responses that activate different metabolic pathways which considerably alter their chemical and physical aspects. On the other hand, insects developed several strategies to overcome plant defense barriers, allowing them to feed, grow and reproduce on their host plants. This review foccuses on several aspects of this complex interaction between plants and insects, including chemical-derived substances, protein-derived molecules and volatile compounds of plants whereas metabolization, sequestration or avoidance are in turn employed by the insects.

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., Author summary The rapid alkalinization factor (RALF) is a small peptide hormone that binds to FERONIA receptor and inhibits cell expansion. The AtRALF1, a root-specific RALF isoform in Arabidopsis, opposes brassinosteroid (BR) effects on root cell elongation. To address the role of AtRALF1 and BRs in regulating cell expansion, we evaluated the response of BR signaling mutants to AtRALF1. We found that the BRI1-associated receptor kinase1 (bak1) mutants are insensitive to AtRALF1 root growth inhibition activity. Although BAK1 showed no effect on the mobilization of Ca2+ and alkalinization responses, it was required for the induction of AtRALF1-responsive genes and for the semi-dwarf phenotype of AtRALF1-overexpressing plants. Different experiments demonstrated that AtRALF1 and BAK1 interact physically in a specific manner. We also suggested that the binding of AtRALF1 to BAK1 is functional because AtRALF1 induces an increase in BAK1 phosphorylation. Our findings indicate that BAK1 plays an important role in AtRALF1 signaling.

Plants respond to pathogens and insect attacks by inducing and accumulating a large set of defense-related proteins. Two homologues of a barley wound-inducible protein (BARWIN) have been characterized in sugarcane, SUGARWIN1 and SUGARWIN2 (sugarcane wound-inducible proteins). Induction of SUGARWINs occurs in response to Diatraea saccharalis damage but not to pathogen infection. In addition, the protein itself does not show any effect on insect development; instead, it has antimicrobial activities toward Fusarium verticillioides, an opportunistic fungus that usually occurs after D. saccharalis borer attacks on sugarcane. In this study, we sought to evaluate the specificity of SUGARWIN2 to better understand its mechanism of action against phytopathogens and the associations between fungi and insects that affect plants. We used Colletotrichum falcatum, a fungus that causes red rot disease in sugarcane fields infested by D. saccharalis, and Ceratocystis paradoxa, which causes pineapple disease in sugarcane. We also tested whether SUGARWIN2 is able to cause cell death in Aspergillus nidulans, a fungus that does not infect sugarcane, and in the model yeast Saccharomyces cerevisiae, which is used for bioethanol production. Recombinant SUGARWIN2 altered C. falcatum morphology by increasing vacuolization, points of fractures and a leak of intracellular material, leading to germling apoptosis. In C. paradoxa, SUGARWIN2 showed increased vacuolization in hyphae but did not kill the fungi. Neither the non-pathogenic fungus A. nidulans nor the yeast S. cerevisiae was affected by recombinant SUGARWIN2, suggesting that the protein is specific to sugarcane opportunistic fungal pathogens.