Marilia Santos Silva

Plant–pathogen interactions: what is proteomics telling us?

Over the years, several studies have been performed to analyse plant–pathogen interactions. Recently, functional genomic strategies, including proteomics and transcriptomics, have contributed to the effort of defining gene and protein function and expression profiles. Using these ‘omic’ approaches, pathogenicity‐ and defence‐related genes and proteins expressed during phytopathogen infections have been identified and enormous datasets have been accumulated. However, the understanding of molecular plant–pathogen interactions is still an intriguing area of investigation. Proteomics has dramatically evolved in the pursuit of large‐scale functional assignment of candidate proteins and, by using this approach, several proteins expressed during phytopathogenic interactions have been identified. In this review, we highlight the proteins expressed during plant–virus, plant–bacterium, plant–fungus and plant–nematode interactions reported in proteomic studies, and discuss these findings considering the advantages and limitations of current proteomic tools.

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

BackgroundHeterologous protein expression in microorganisms may contribute to identify and demonstrate antifungal activity of novel proteins. The Solanum nigrum osmotin-like protein (SnOLP) gene encodes a member of pathogenesis-related (PR) proteins, from the PR-5 sub-group, the last comprising several proteins with different functions, including antifungal activity. Based on deduced amino acid sequence of SnOLP, computer modeling produced a tertiary structure which is indicative of antifungal activity.ResultsTo validate the potential antifungal activity of SnOLP, a hexahistidine-tagged mature SnOLP form was overexpressed in Escherichia coli M15 strain carried out by a pQE30 vector construction. The urea solubilized His6-tagged mature SnOLP protein was affinity-purified by immobilized-metal (Ni2+) affinity column chromatography. As SnOLP requires the correct formation of eight disulfide bonds, not correctly formed in bacterial cells, we adapted an in vitro method to refold the E. coli expressed SnOLP by using reduced:oxidized gluthatione redox buffer. This method generated biologically active conformations of the recombinant mature SnOLP, which exerted antifungal action towards plant pathogenic fungi (Fusarium solani f. sp.glycines, Colletotrichum spp., Macrophomina phaseolina) and oomycete (Phytophthora nicotiana var. parasitica) under in vitro conditions.ConclusionSince SnOLP displays activity against economically important plant pathogenic fungi and oomycete, it represents a novel PR-5 protein with promising utility for biotechnological applications.

A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination

BackgroundAsian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris.ResultsA cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%.ConclusionsOur data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.

Variant Cry1Ia toxins generated by DNA shuffling are active against sugarcane giant borer.

Sugarcane giant borer (Telchin licus licus) is a serious sugarcane pest in Americas whose endophytic lifestyle hampers effective chemical and biological controls. Therefore, development of alternative control methods is extremely important. Envisaging development of transgenic plants resistant to this pest, we investigated the effect of the Bacillus thuringiensis Cry protein Cry1Ia12synth (truncated protein lacking C-terminus with plant codon usage) and variants against T. l. licus. cry1Ia12synth gene was used to generate mutated variants, which were screened for toxicity toward T. l. licus. For that purpose, an innovative technique combining cry gene shuffling with phage-display was used to build a combinatorial library comprising 1.97x10(5) Cry1Ia12synth variants. Screening of this library for variants binding to T. l. licus Brush Border Midgut Vesicles led to the identification of hundreds of clones, out of which 30 were randomly chosen for toxicity testing. Bioassays revealed four variants exhibiting activity against T. l. licus as compared to the non-toxic Cry1Ia12synth. Eight single substitutions sites were found in these active variants. Based on theoretical molecular modelling, the probable implications of these mutations are discussed. Therefore, we have four genes encoding Cry1Ia12synth variants active against T. l. licus promising for future development of resistant transgenic sugarcane lines.

Divergência genética entre acessos açucarados e não açucarados de mandioca

The objective of this work was to estimate the genetic divergence among sugary and nonsugary cassava accessions, through molecular markers, and quantitative and qualitative characters, as well as to determine the correlation among these estimates. Four sugary cassava accessions and four nonsugary ones were used, including two landraces and two nonsugary improved varieties. The accessions were evaluated in field conditions and in laboratories, with RAPD markers, in 12 quantitative and 33 qualitative characters. Matrixes of genetic dissimilarity/distance among accessions were estimated, based on qualitative and quantitative characters, molecular markers, and on the correlation significance among matrixes. High genetic divergence among the evaluated accessions was observed, the sugary accessions were differentiated from the landraces and improved nonsugary varieties. The distances estimated through molecular markers and qualitative characters showed the high association among each other and moderate association with the distance estimated through quantitative characters.

Análise da recuperação do genitor recorrente em maracujazeiro-azedo por meio de marcadores RAPD

Brazil is the largest world producer of passion fruit, however, it has been observed a reduction in the productivity in recent years due, mainly, to phytosanitary factors. At Embrapa Cerrados, the transfer of resistance genes from wild to commercial species of passion fruit has been made through interspecific hybridations, followed by a backcrossing molecular marker-assisted program. The objective this work was to verify the recovery of recurrent genome at the plants RC4 and RC5 [(Passiflora edulis x Passiflora setacea) x Passiflora edulis] based on RAPD markers. The study was developed at Embrapa Cerrados Laboratory of Genetics and Molecular Biology. DNA samples of each genetic material (17 RC4 plants, 16 RC5 plants, Passiflora edulis and Passiflora setacea) were amplified to obtain RAPD markers. There were used 12 decamer primers for plants RC4 and 14 decamer primers for plants RC5. The RAPD markers generated were converted into a matrix of binary data. There were a high percentage of polymorphic markers as a result of interspecific base crossing. The smallest genetic similarity was observed between species P. edulis and P. setacea, highlighting the large genetic distance of these commercial and wild varieties, respectively.

Characterization of sweet cassava accessions based on molecular, quantitative and qualitative data

The purpose of this study was to estimate the genetic divergence in sweet cassava accessions by molecular markers and quantitative and qualitative characters, as well as determine the correlation between these estimates. Sixteen sweet cassava accessions of the Regional Cassava Germplasm Bank of the Cerrado were evaluated under field conditions, for 13 quantitative and 33 qualitative characters. In the laboratory, the accessions were evaluated with RAPD markers. Subsequently, matrixes of genetic dissimilarity/distance among the accessions were estimated based on molecular markers and quantitative and qualitative characters. Besides, the significance of the correlation between the matrixes was estimated. The RAPD, qualitative and quantitative data indicated the existence of high divergence among the accessions. The divergences estimated by molecular markers and by quantitative traits were weakly associated with each other and moderately with the divergence estimated by qualitative characters.

Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil

Summary Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination‐related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR‐based 2−ΔΔCt analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g−1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g−1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

Genes associated with hypersensitive response (HR) in the citrus EST database (CitEST)

Plants are continuously exposed to pathogen attack, but successful infection is rare because they protect themselves against pathogens using a wide range of response mechanisms. One of them is the hypersensitive response (HR), which is a form of cell death often associated with plant resistance to pathogen infection to prevent the spreadsebpg@cnpq.br sebpg@cnpq.br of the potential pathogen from infected to uninfected tissues. Cell death is activated by recognition of pathogen-derived molecules by the resistance (R) gene products, and is associated with the massive accumulation of reactive oxygen species (ROS), salicylic acid (SA), and other pro-death signals such as nitric oxide (NO). The analysis of the citrus EST (CitEST) database revealed the presence of putative genes likely to be involved in HR through their products, like metacaspases, lipoxygenases, phospholipases, pathogenesis-related proteins, glutathione transferases/peroxidases, enzymes involved in the phenylpropanoid pathway and in the formation and detoxification of ROS, as well as those involved in the formation and regulation of ion channels, SA and NO. By analysis of the EST database of Citrus, it was possible to identify several putative genes that code for key enzymes involved in HR triggering and also in plant defense against biotic and abiotic stress.

Pre-germination treatments and storage of cassava seeds and their correlation with emergence of seedlings 1

Despite propagation of the cassava crop be done by cuttings for commercial purposes, the majority of accesses keeps active the sexual propagation system; what is essential to genetic breeding programs. Cassava seeds, however, have low and uneven germination rates. Considering such event, the objective of this study was to evaluate the efficiency of the storage, and different pre-germination treatments, on cassava seedling emergence. For this, an experiment was carried out with seeds obtained by open pollination between plants of four sweet cassava cultivars, with 10 pre-germination treatments; in addition to a control treatment. The viability of seeds subjected to different treatments was assessed by tetrazolium test; and the assessment of seedling emergence was performed by daily counts of the number of emerged seedlings. Data obtained were expressed in: emergence percentage; emergence speed index; and mean time of seedling emergence. It was concluded that storage, at 4 °C, during one year, increases seed emergence percentage and favors cassava seedling emergence speed; and that the dry heat (60 oC), during seven and 14 days, also favors their emergence speed .