Marcio C. Silva-Filho

Plant-insect interactions: an evolutionary arms race between two distinct defense mechanisms

Nesta revisao, a interacao planta-inseto e abordada como um sistema dinâmico, sujeito a continuas variacoes e mudancas. As plantas desenvolveram diferentes mecanismos para reduzir o ataque de insetos, incluindo respostas especificas que ativam diferentes vias metabolicas as quais alteram consideravelmente suas caracteristicas quimicas e fisicas. Por outro lado, os insetos desenvolveram varias estrategias para superar as barreiras defensivas das plantas, permitindo a sua alimentacao, desenvolvimento e reproducao em seus hospedeiros. Esta revisao enfoca varios aspectos desta complexa interacao entre plantas e insetos, incluindo substâncias derivadas de compostos quimicos, moleculas produzidas a partir do processamento de proteinas e compostos volateis das plantas, enquanto que a metabolizacao, sequestro ou fuga sao empregados em contrapartida pelos insetos.

Signal transduction-related responses to phytohormones and environmental challenges in sugarcane

BackgroundSugarcane is an increasingly economically and environmentally important C4 grass, used for the production of sugar and bioethanol, a low-carbon emission fuel. Sugarcane originated from crosses of Saccharum species and is noted for its unique capacity to accumulate high amounts of sucrose in its stems. Environmental stresses limit enormously sugarcane productivity worldwide. To investigate transcriptome changes in response to environmental inputs that alter yield we used cDNA microarrays to profile expression of 1,545 genes in plants submitted to drought, phosphate starvation, herbivory and N2-fixing endophytic bacteria. We also investigated the response to phytohormones (abscisic acid and methyl jasmonate). The arrayed elements correspond mostly to genes involved in signal transduction, hormone biosynthesis, transcription factors, novel genes and genes corresponding to unknown proteins.ResultsAdopting an outliers searching method 179 genes with strikingly different expression levels were identified as differentially expressed in at least one of the treatments analysed. Self Organizing Maps were used to cluster the expression profiles of 695 genes that showed a highly correlated expression pattern among replicates. The expression data for 22 genes was evaluated for 36 experimental data points by quantitative RT-PCR indicating a validation rate of 80.5% using three biological experimental replicates. The SUCAST Database was created that provides public access to the data described in this work, linked to tissue expression profiling and the SUCAST gene category and sequence analysis. The SUCAST database also includes a categorization of the sugarcane kinome based on a phylogenetic grouping that included 182 undefined kinases.ConclusionAn extensive study on the sugarcane transcriptome was performed. Sugarcane genes responsive to phytohormones and to challenges sugarcane commonly deals with in the field were identified. Additionally, the protein kinases were annotated based on a phylogenetic approach. The experimental design and statistical analysis applied proved robust to unravel genes associated with a diverse array of conditions attributing novel functions to previously unknown or undefined genes. The data consolidated in the SUCAST database resource can guide further studies and be useful for the development of improved sugarcane varieties.

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin (M(r) 26000) and four trypsins (T1-T4) with the following properties: T1, K(m) 0.3 microM, M(r) 70000; T2, K(m) 0.4 microM, M(r) 67000; T3, K(m) 2.4 microM, M(r) 29000; T4, K(m) 15 microM, M(r) 17000. Diet-fed larvae have a chymotrypsin (M(r) 26000) and a major trypsin (K(m) 2.9 microM, M(r) 29000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K(m) values) and a putative decreased affinity for PIs.

Effects of soybean proteinase inhibitor on development, survival and reproductive potential of the sugarcane borer, Diatraea saccharalis

One approach that can be employed in integrated pest management is the use of proteins with antinutritional effects on insect metabolism and development. The antimetabolic properties of soybean proteinase inhibitor (SPI) on growth of neonate larvae of the sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) have been evaluated. When incorporated into an artificial diet at 0.5% (w/w), SPI retarded growth rate and development of larvae when compared with larvae fed on artificial diet alone. However, larval survival was not significantly affected. The purpose of our research was to calculate demographic statistics for the sugarcane borer reared on diet either with or without semi‐purified extract of SPI. Net reproductive rate (R0), instantaneous rate of increase (rm), combined age‐specific survivorship (lx) and age specific fecundity (mx) provide information about population growth potential. These parameters were measured in order to determine the effects of the proteinase inhibitor on the insects population dynamics. The observed differences would potentially translate into large reductions in population growth, indicating a potential value of using SPI for protecting sugarcane plants against damage by the sugarcane borer.

AtPIN: Arabidopsis thaliana Protein Interaction Network

BackgroundProtein-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.DescriptionAll 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.ConclusionsAtPIN 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.

Sugarcane Functional Genomics: Gene Discovery for Agronomic Trait Development

Sugarcane is a highly productive crop used for centuries as the main source of sugar and recently to produce ethanol, a renewable bio-fuel energy source. There is increased interest in this crop due to the impending need to decrease fossil fuel usage. Sugarcane has a highly polyploid genome. Expressed sequence tag (EST) sequencing has significantly contributed to gene discovery and expression studies used to associate function with sugarcane genes. A significant amount of data exists on regulatory events controlling responses to herbivory, drought, and phosphate deficiency, which cause important constraints on yield and on endophytic bacteria, which are highly beneficial. The means to reduce drought, phosphate deficiency, and herbivory by the sugarcane borer have a negative impact on the environment. Improved tolerance for these constraints is being sought. Sugarcanes ability to accumulate sucrose up to 16% of its culm dry weight is a challenge for genetic manipulation. Genome-based technology such as cDNA microarray data indicates genes associated with sugar content that may be used to develop new varieties improved for sucrose content or for traits that restrict the expansion of the cultivated land. The genes can also be used as molecular markers of agronomic traits in traditional breeding programs.

Changes in midgut endopeptidase activity of Spodoptera frugiperda (Lepidoptera: Noctuidae) are responsible for adaptation to soybean proteinase inhibitors.

Abstract The development of transgenic maize plants expressing soybean proteinase inhibitors could reduce the economic damage of one of the major maize pests in Brazil, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797). We examined the influence of soybean proteinase inhibitors on digestive enzyme properties and development of S. frugiperda larvae. The inhibition of trypsin and chymotrypsin activities in vitro by soybean proteinase inhibitors suggested that either Kunitz (SBTI) or Bowman-Birk (SBBI) would have a potential antimetabolic effect when ingested by insect larvae. However, chronic ingestion of semipurified soybean inhibitors did not result in a significant reduction of growth and development of fall armyworm. Therefore, digestive serine proteinase activities (trypsin and chymotrypsin) of fall armyworm larvae were characterized. The results suggest that S. frugiperda was able to physiologically adapt to dietary proteinase inhibitors by altering the complement of proteolytic enzymes in the insect midguts.

Differential usage of two in-frame translational start codons regulates subcellular localization of Arabidopsis thaliana THI1

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 Kozaks consensus. It suggests leaky ribosomal scanning, reinitiation or the internal entry of ribosomes to assure mitochondrial protein import.

Molecular evolution of Bowman-Birk type proteinase inhibitors in flowering plants

The Bowman-Birk family (BBI) of proteinase inhibitors is probably the most studied family of plant inhibitors. We describe the primary structure and the gene expression profile of 14 putative BBIs from the sugarcane expressed sequence tag database and show how we used these newly discovered sequences together with 87 previously described BBI sequences from the GenBank database to construct phylogenetic trees for the BBI family. Phylogenetic analysis revealed that BBI-type inhibitors from monocotyledonous and dicotyledonous plants could be clearly separated into different groups, while the overall topology of the BBI tree suggests a different pattern of evolution for BBI families in flowering plants. We also found that BBI proteinase inhibitors from dicotyledonous plants were well conserved, accumulating only slight differences during their evolution. In addition, we found that BBIs from monocotyledonous plants were highly variable, indicating an interesting process of evolution based on internal gene duplications and mutation events.

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.