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.

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.

Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple.

The Rapid Alkalinization Factor (RALF) is a plant hormone peptide that inhibits proton transport causing alkalinization of the extracellular media. To detect the alkalinization response elicited by RALF peptides in root cells, Arabidopsis seedlings are carefully transferred to a gel containing the pH-sensitive indicator bromocresol purple, treated with the peptide and photographed after 30 min. Herein the protocol is optimized for evaluation of exogenous treatment, described in detail and expected results are presented.

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.

Structural and Functional Characterization of PR-4 SUGARWINs From Sugarcaneand Their Role in Plant Defense.

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.

Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling.

In flowering plants, fertilization requires complex cell-to-cell communication events between the pollen tube and the female reproductive tissues, which are controlled by extracellular signaling molecules interacting with receptors at the pollen tube surface. We found that two such receptors in Arabidopsis, BUPS1 and BUPS2, and their peptide ligands, RALF4 and RALF19, are pollen tube-expressed and are required to maintain pollen tube integrity. BUPS1 and BUPS2 interact with receptors ANXUR1 and ANXUR2 via their ectodomains, and both sets of receptors bind RALF4 and RALF19. These receptor-ligand interactions are in competition with the female-derived ligand RALF34, which induces pollen tube bursting at nanomolar concentrations. We propose that RALF34 replaces RALF4 and RALF19 at the interface of pollen tube-female gametophyte contact, thereby deregulating BUPS-ANXUR signaling and in turn leading to pollen tube rupture and sperm release.

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.

Plant-insect-pathogen interactions: a naturally complex ménage à trois.

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.

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.

Comparative analysis of expression profiling of the trypsin and chymotrypsin genes from Lepidoptera species with different levels of sensitivity to soybean peptidase inhibitors.

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.

Antagonistic relationship between AtRALF1 and brassinosteroid regulates cell expansion-related genes.

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 sugarcane defense protein SUGARWIN2 causes cell death in Colletotrichum falcatum but not in non-pathogenic fungi.

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.

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.

Biological activity of nine recombinant AtRALF peptides: implications for their perception and function in Arabidopsis.

RALF is a small (5 kDa) and ubiquitous plant peptide signal. It was first isolated from tobacco leaf protein extracts owing to its capacity to alkalinize the extracellular media of cell suspensions. RALFs inhibit root growth and hypocotyl elongation, and a role for RALFs in cell expansion has also been proposed. Arabidopsis has 37 RALF isoforms (AtRALF), but only a small group of nine has high primary structure identity to the original RALF peptide isolated from tobacco. Herein, we report the heterologous production of these nine peptides in Escherichia coli and the evaluation of their activity in five biological assays. All AtRALF peptides produced showed strong alkalinizing activities, with the exception of the pollen-specific isoform AtRALF4. Although it exhibited no inhibitory activity in the root growth and hypocotyl elongation assays, AtRALF4 is a strong inhibitor of pollen germination. Our data demonstrate that the divergence in the tissue specificity and gene expression patterns of the different AtRALFs does not change the fact that their main role seems to be the regulation of cell expansion. Furthermore, different activities in the alkalinization assays upon the addition of two consecutive and saturating doses of the peptides suggest that the peptides are likely being sensed by specific receptors.

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.

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.

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.

General up regulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor.

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.