Antisense oligonucleotide as a new technology application for CsLOB1 gene silencing aiming citrus canker resistance.

Citrus canker disease, caused by Xanthomonas citri subsp. citri, poses a significant threat to global citrus production. The control of the disease in the field relies mainly on the use of conventional tools such as copper compounds, which are harmful to the environment and could lead to bacterial resistance. This scenario stresses the need for new and sustainable technologies to control phytopathogens, representing a key challenge in developing studies that translate basic into applied knowledge. During infection, X. citri subsp. citri secretes a transcriptional activator-like effector that enters the nucleus of plant cells, activating the expression of the canker susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1). In this study, we explored the use of antisense oligonucleotides (ASOs) with phosphorothioate modifications to transiently inhibit the gene expression of CsLOB1 in Citrus sinensis. We designed and validated three potential ASO sequences, which led to a significant reduction in disease symptoms compared to the control. The selected ASO3-CsLOB1 significantly decreased the expression level of CsLOB1 when delivered through two distinct delivery methods and the reduction of the symptoms ranged from approximately 15% to 83%. Notably, plants treated with ASO3 did not exhibit an increase in symptoms development over the evaluation period. This study highlights the efficacy of ASO technology, based on short oligonucleotide chemically modified sequences, as a promising tool for controlling phytopathogens without the need for genetic transformation or plant regeneration. Our results demonstrate the potential of ASOs as a biotechnological tool for the management of citrus canker disease.

Repurposing streptomycin and chloramphenicol against bacterial pathogens by combination with diminazene aceturate.

Bacterial resistance is a threat to health worldwide, mainly due to reduced effective treatment. In this context, the search for strategies to control such infections and suppress antimicrobial resistance is necessary. One of the strategies that has been used is combination therapy. In the present work, we investigated the in vitro efficacy of the antimicrobials diminazene aceturate (DA), chloramphenicol (CHL), and streptomycin (STP) alone and in combination against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus clinical isolates. DA was capable of inhibiting all strains with MIC of 25-400 µg mL-1, while STP and CHL showed antibacterial activity with minimum inhibitory concentration (MICs) of ≤3.12-400 µg mL-1. The combination of aceturate with STP showed synergism toward almost all Gram-negative bacteria, with fractional inhibitory concentration index (FICIs) of 0.09-0.37. In addition, for CHL and aceturate, synergisms for Gram-negative and -positive strains were observed. A time-kill assay against E. coli revealed that the aceturate and STP combination can inhibit bacterial growth in a shorter time when compared with single antibiotics. In addition, antimicrobials did not show hemolytic activity even at the highest concentrations used. Therefore, the antimicrobial combinations presented in this work showed important results, demonstrating that combined therapy can be used as an alternative strategy for pathogen control.

Seasonal differences in seminal plasma proteins from two bovine breeds adapted to a subtropical climate.

This study was designed to evaluate the seasonal expression of seminal plasma proteins from two bovine breeds adapted to a subtropical climate and their associations with post-thawing sperm and environmental characteristics. Semen samples were obtained three times in summer and three times in winter from four Crioulo Lageano and four Angus bulls. Seminal plasma was obtained by centrifugation, and the other portion of the semen was cryopreserved. Seminal plasma proteins were identified by 2D-nanoUPLC-MSE. Post-thawing assessments of sperm kinetics, morphology and membrane integrity were performed. Environmental data such as air temperature, air humidity and black globe temperature (BGT) were recorded, and the temperature-humidity index (THI) was calculated in summer and winter. Results showed that the climate varied significantly between seasons. Although no statistical differences were observed in semen quality between breeds, the protein profiles varied within and between seasons. We suggest that the most critical proteins in summer affecting sperm characteristics were TIMP-2, DNase, Clusterin, CFAH and GPx6. TIMP-2 and DNase showed a higher abundance in Crioulo Lageano in comparison with Angus, while Clusterin, CFAH and GPx6 presented a lower abundance. To the best of our knowledge, this is the first report of a recently evolved type of glutathione peroxidase, GPx6, in seminal plasma of bovines. In winter, five proteins were considered to be more critical: BSP1, BSP3, CCL2, Sulfhydryl oxidase and TIMP-2. BSP1 and TIMP-2 showed a lower abundance while BSP3, CCL2 and Sulfhydryl oxidase presented a higher abundance in this season in Crioulo Lageano in comparison with Angus.

RESUMO: Este estudo foi desenvolvido para avaliar a expressão sazonal de proteínas plasmáticas seminais de duas raças bovinas adaptadas ao clima subtropical e suas associações com espermatozóides pós-descongelamento e características ambientais. Amostras de sêmen foram obtidas três vezes no verão e três no inverno de quatro touros Crioulo Lageano e quatro Angus. O plasma seminal foi obtido por centrifugação e outra porção do sêmen foi criopreservada. As proteínas plasmáticas seminais foram identificadas por 2D-nanoUPLC-MSE. Foram realizadas avaliações pós-descongelamento da cinética espermática, morfologia e integridade da membrana. Dados ambientais como temperatura do ar, umidade do ar e temperatura do globo negro (BGT) foram registrados, e o índice temperatura-umidade (THI) foi calculado no verão e no inverno. Os resultados mostraram que o clima variou significativamente entre as estações. Embora não tenham sido observadas diferenças estatísticas na qualidade do sêmen entre as raças, os perfis proteicos variaram dentro e entre as estações. Sugerimos que as proteínas mais críticas no verão que afetam as características espermáticas foram TIMP-2, DNase, Clusterin, CFAH e GPx6. TIMP-2 e DNase apresentaram maior abundância em Crioulo Lageano em comparação com Angus, enquanto Clusterin, CFAH e GPx6 apresentaram menor abundância. Até onde sabemos, este é o primeiro relato de um tipo recentemente desenvolvido de glutationa peroxidase, GPx6, no plasma seminal de bovinos. No inverno, cinco proteínas foram consideradas mais críticas: BSP1, BSP3, CCL2, sulfidril oxidase e TIMP-2. BSP1 e TIMP-2 apresentaram menor abundância, enquanto BSP3, CCL2 e Sulfidril oxidase apresentaram maior abundância nesta temporada em Crioulo Lageano em comparação com Angus.

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence.

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra-pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc - Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max-exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT-PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.

Quantitative expression of microRNAs in Brassica oleracea infected with Xanthomonas campestris pv. campestris.

Brassica oleracea var. capitata (cabbage) is an economically important crop affected by black rot disease caused by Xanthomonas campestris pv. campestris (Xcc). MicroRNAs (miRNAs) play an important role in plant defense modulation and therefore the analysis of these molecules can help better understand plant-pathogen interactions. In this study, we report the differential expression of four miRNAs that seem to participate in the plant response to Xcc infection. Northern Blot and RT-qPCR techniques were used to measure miRNA expression in resistant (União) and susceptible (Kenzan) cultivars. From 6 miRNAs analyzed, 4 were detected and differentially expressed, showing a down- and upregulated expression profile in susceptible and resistant cultivars, respectively. These results suggest that miR156, miR167, miR169, and miR390 could play a role in B. oleracea resistance enhancement against Xcc and could be explored as potential resistance markers in B. oleracea-Xcc interaction.

Mechanism of the drought tolerance of a transgenic soybean overexpressing the molecular chaperone BiP.

Drought is one of major constraints that limits agricultural productivity. Some factors, including climate changes and acreage expansion, indicates towards the need for developing drought tolerant genotypes. In addition to its protective role against endoplasmic reticulum (ER) stress, we have previously shown that the molecular chaperone binding protein (BiP) is involved in the response to osmotic stress and promotes drought tolerance. Here, we analyzed the proteomic and metabolic profiles of BiP-overexpressing transgenic soybean plants and the corresponding untransformed line under drought conditions by 2DE-MS and GC/MS. The transgenic plant showed lower levels of the abscisic acid and jasmonic acid as compared to untransformed plants both in irrigated and non-irrigated conditions. In contrast, the level of salicylic acid was higher in transgenic lines than in untransformed line, which was consistent with the antagonistic responses mediated by these phytohormones. The transgenic plants displayed a higher abundance of photosynthesis-related proteins, which gave credence to the hypothesis that these transgenic plants could survive under drought conditions due to their genetic modification and altered physiology. The proteins involved in pathways related to respiration, glycolysis and oxidative stress were not signifcantly changed in transgenic plants as compared to untransformed genotype, which indicate a lower metabolic perturbation under drought of the engineered genotype. The transgenic plants may have adopted a mechanism of drought tolerance by accumulating osmotically active solutes in the cell. As evidenced by the metabolic profiles, the accumulation of nine primary amino acids by protein degradation maintained the cellular turgor in the transgenic genotype under drought conditions. Thus, this mechanism of protection may cause the physiological activities including photosynthesis to be active under drought conditions.

Differential accumulation of Xanthomonas campestris pv. campestris proteins during the interaction with the host plant: Contributions of an in vivo system.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot, a highly destructive disease that affects all brassicas. This work aimed to study the interaction Xcc-Brassica oleracea using an in vivo system in an attempt to identify proteins involved in pathogenicity. We used label-free shotgun 2D-nanoUPLC/MSE to analyze Xcc proteins in three conditions: in the interaction with susceptible (REK) and resistant (REU) plants and in culture medium (control condition). A model of Xcc-susceptible host interaction is proposed and shows that Xcc increases the abundance of several crucial proteins for infection and cell protection. In this study, we also confirmed the differential expression by qPCR analysis of selected genes. This is the first report showing a large-scale identification of proteins in an in vivo host plant condition. Considering that most studies involving phytopathogens are in vitro (growth in culture medium or in plant extract), this work contributes with relevant information related to the plant-pathogen interaction in planta.

A year (2014-2015) of plants in Proteomics journal. Progress in wet and dry methodologies, moving from protein catalogs, and the view of classic plant biochemists.

The present review is an update of the previous one published in Proteomics 2015 Reviews special issue [Jorrin-Novo, J. V. et al., Proteomics 2015, 15, 1089-1112] covering the July 2014-2015 period. It has been written on the bases of the publications that appeared in Proteomics journal during that period and the most relevant ones that have been published in other high-impact journals. Methodological advances and the contribution of the field to the knowledge of plant biology processes and its translation to agroforestry and environmental sectors will be discussed. This review has been organized in four blocks, with a starting general introduction (literature survey) followed by sections focusing on the methodology (in vitro, in vivo, wet, and dry), proteomics integration with other approaches (systems biology and proteogenomics), biological information, and knowledge (cell communication, receptors, and signaling), ending with a brief mention of some other biological and translational topics to which proteomics has made some contribution.

Cowpea-Meloidogyne incognita interaction: Root proteomic analysis during early stages of nematode infection.

Cowpea (Vigna unguiculata L. Walp) is an important legume species well adapted to low fertility soils and prolonged drought periods. One of the main problems that cause severe yield losses in cowpea is the root-knot nematode Meloidogyne incognita. The aim of this work was to analyze the differential expression of proteins in the contrasting cultivars of cowpea CE 31 (highly resistant) and CE 109 (slightly resistant) during early stages of M. incognita infection. Cowpea roots were collected at 3, 6, and 9 days after inoculation and used for protein extraction and 2-DE analysis. From a total of 59 differential spots, 37 proteins were identified, mostly involved in plant defense, such as spermidine synthase, patatin, proteasome component, and nitrile-specifier protein. A follow-up study was performed by quantitative RT-PCR analysis of nine selected proteins and the results revealed a very similar upregulation trend between the protein expression profiles and the corresponding transcripts. This study also identified ACT and GAPDH as a good combination of reference genes for quantitative RT-PCR analysis of the pathosystem cowpea/nematode. Additionally, an interactome analysis showed three major pathways affected by nematode infection: proteasome endopeptidase complex, oxidative phosphorylation, and flavonoid biosynthesis. Taken together, the results obtained by proteome, transcriptome, and interactome approaches suggest that oxidative stress, ubiquitination, and glucosinolate degradation may be part of cowpea CE 31 resistance mechanisms in response to nematode infection.

Differential expression of genes potentially related to the callogenesis and in situ hybridization of SERK gene in macaw palm (Acrocomia aculeata Jacq.) Lodd. ex Mart.

For the purpose of understanding the molecular processes triggered during callus formation in macaw palm, the expression of seven genes potentially involved in this process, identified in previous studies and from the literature, was investigated by RT-qPCR. In addition, in situ hybridization of the SERK gene was performed. Leaf tissues from adult plants from two macaw palm accession were inoculated in a medium combined with Picloram at a concentration of 450 µM to induce callus. The expression analysis was performed from leaf samples from two accessions of different origins (Municipalities of Tiros, MG, and Buriti Vermelho, DF, Brazil), which are characterized as non-responsive (NR) and responsive (R), respectively. The material was collected before callus induction (0 DAI, initial day) and 120 days after callus induction (120 DAI). Genes related to development (SERK, OASA, EF1, ANN1) and stress (LEA, CAT2, and MDAR5) were evaluated. The results obtained showed that all the genes involved with the development had their expressions downregulated at 0 DAI when the accession R was compared with the accession NR. On the other hand, it was possible to observe that these genes were upregulated at 120 DAI. The LEA stress gene showed a tendency to increase expression in the NR accession, while the R accession showed decreased expression and the CAT2 and MDAR5 genes showed upregulation in both accessions. In situ hybridization showed SERK transcripts in the vascular bundles, indicating the expression of SERK in this region, in addition to its expression in calluses. The results obtained in this study support our hypothesis that the regulation of genes involved in the control of oxidative stress and development is crucial for the formation of calluses in macaw palm.

Identification of host proteins modulated by the virulence factor AC2 of Tomato chlorotic mottle virus in Nicotiana benthamiana.

Tomato, one of the most important crops cultivated worldwide, has been severely affected by begomoviruses such as the Tomato chlorotic mottle virus (ToCMoV). Virulence factor AC2 is considered crucial for a successful virus-plant interaction and is known to act as a transcriptional activator and in some begomoviruses to function as an RNA silencing suppressor factor. However, the exact functions of the AC2 protein of the begomovirus ToCMoV are not yet established. The aim of the present study was to identify differentially expressed proteins of the model plant Nicotiana benthamiana in response to the expression of the AC2 gene, isolated from ToCMoV. N. benthamiana plants were inoculated with Agrobacterium tumefaciens containing the viral vector Potato virus X (PVX) and with the PVX-AC2 construction. 2DE was performed and proteins were identified by MS. The results showed that the expression of ToCMoV AC2 alters the levels of several host proteins, which are important for normal plant development, causing an imbalance in cellular homeostasis. This study highlights the effect of AC2 in the modulation of plant defense processes by increasing the expression of several oxidative stress-related and pathogenesis-related proteins, as well as its role in modulating the proteome of the photosynthesis and energy production systems.

Effects of acute exercise over heart proteome from monogenic obese (ob/ob) mice.

Exercise is recognized to prevent and attenuate several metabolic and cardiovascular disorders. Obesity is commonly related to cardiovascular diseases, frequently resulting in heart failure and death. To elucidate the effects of acute exercise in heart tissue from obese animals, 12-week-old C57BL6/J obese (ob/ob) and non-obese (ob/OB) mice were submitted to a single bout of swimming and had their hearts analyzed by proteomic techniques. Mice were divided into three groups: control (ob/ob, n = 3; ob/OB, n = 3); a moderate intensity consisting of 20 min of swimming around 90% of Maximal Lactate Steady State (ob/ob, n = 3; ob/OB, n = 3), and a high intensity exercise performed as an incremental overload test (ob/ob, n = 3; ob/OB, n = 3). Obesity modulations were analyzed by comparing ob/ob and ob/OB control groups. Differential 2-DE analysis revealed that single session of exercise was able to up-regulate: myoglobin (ob/ob), aspartate aminotransferase (ob/OB) and zinc finger protein (ob/OB) and down-regulate: nucleoside diphosphate kinase B (ob/OB), mitochondrial aconitase (ob/ob and ob/OB) and fatty acid binding protein (ob/ob). Zinc finger protein and α-actin were up-regulated by the effect of obesity on heart proteome. These data demonstrate the immediate response of metabolic and stress-related proteins after exercise so as contractile protein by obesity modulation on heart proteome.

Proteomic Insights of Cowpea Response to Combined Biotic and Abiotic Stresses.

The co-occurrence of biotic and abiotic stresses in agricultural areas severely affects crop performance and productivity. Drought is one of the most adverse environmental stresses, and its association with root-knot nematodes further limits the development of several economically important crops, such as cowpea. Plant responses to combined stresses are complex and require novel adaptive mechanisms through the induction of specific biotic and abiotic signaling pathways. Therefore, the present work aimed to identify proteins involved in the resistance of cowpea to nematode and drought stresses individually and combined. We used the genotype CE 31, which is resistant to the root-knot nematode Meloidogyne spp. And tolerant to drought. Three biological replicates of roots and shoots were submitted to protein extraction, and the peptides were evaluated by LC-MS/MS. Shotgun proteomics revealed 2345 proteins, of which 1040 were differentially abundant. Proteins involved in essential biological processes, such as transcriptional regulation, cell signaling, oxidative processes, and photosynthesis, were identified. However, the main defense strategies in cowpea against cross-stress are focused on the regulation of hormonal signaling, the intense production of pathogenesis-related proteins, and the downregulation of photosynthetic activity. These are key processes that can culminate in the adaptation of cowpea challenged by multiple stresses. Furthermore, the candidate proteins identified in this study will strongly contribute to cowpea genetic improvement programs.

A Capsid Protein Fragment of a Fusagra-like Virus Found in Carica papaya Latex Interacts with the 50S Ribosomal Protein L17.

Papaya sticky disease is caused by the association of a fusagra-like and an umbra-like virus, named papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), respectively. Both viral genomes are encapsidated in particles formed by the PMeV ORF1 product, which has the potential to encode a protein with 1563 amino acids (aa). However, the structural components of the viral capsid are unknown. To characterize the structural proteins of PMeV and PMeV2, virions were purified from Carica papaya latex. SDS-PAGE analysis of purified virus revealed two major proteins of ~40 kDa and ~55 kDa. Amino-terminal sequencing of the ~55 kDa protein and LC-MS/MS of purified virions indicated that this protein starts at aa 263 of the deduced ORF1 product as a result of either degradation or proteolytic processing. A yeast two-hybrid assay was used to identify Arabidopsis proteins interacting with two PMeV ORF1 product fragments (aa 321-670 and 961-1200). The 50S ribosomal protein L17 (AtRPL17) was identified as potentially associated with modulated translation-related proteins. In plant cells, AtRPL17 co-localized and interacted with the PMeV ORF1 fragments. These findings support the hypothesis that the interaction between PMeV/PMeV2 structural proteins and RPL17 is important for virus-host interactions.

Comparative proteomics between natural Microcystis isolates with a focus on microcystin synthesis.

BACKGROUND: Microcystis aeruginosa is a species of cyanobacteria commonly found in a number of countries and frequently related to animal poisoning episodes due to its capacity to produce the cyanotoxin known as microcystin. Despite vast literature on microcystin structures and their deleterious effects, little is known about its synthesis by cyanobacteria. Therefore, this study used proteomic tools to compare two M. aeruginosa strains, contrasting them for microcystin production. RESULTS: 2-DE gels were performed and 30 differential protein spots were chosen. Among them, 11 protein spots were unique in the toxin producing strain and 8 in the non-toxin producing strain, and 14 protein spots were shown on both 2-DE gels but expressed differently in intensity. Around 57% of the tandem mass spectrometry identified proteins were related to energy metabolism, with these proteins being up-regulated in the toxin producing strain. CONCLUSIONS: These data suggest that the presence of higher quantities of metabolic enzymes could be related to microcystin metabolism in comparison to the non-toxin producing strain. Moreover, it was suggested that the production of microcystin could also be related to other proteins than those directly involved in its production, such as the enzymes involved in the Calvin cycle and glycolysis.

Priming of defense-related genes in Brassica oleracea var. capitata using concentrated metabolites produced by Rhizobium tropici CIAT 899.

To verify the potential of metabolites extracted from Rhizobium tropici to trigger the priming of defense responses in cruciferous plants, we analyzed the expression of defense-related genes by qRT-PCR. Brassica oleracea var. capitata, susceptible to Xanthomonas campestris pv. campestris, were grown in greenhouse conditions. At 18 days after sowing, plants were inoculated with 1 mL of 1% concentrated metabolites produced by R. tropici (CM-RT) in the root. In a second experiment, leaves were sprayed with 1 mL of a solution containing 1% CM-RT. Aerial and root tissue were collected separately at 0 (non-treated control condition), 24, and 48 h after application, submitted to RNA extraction and gene expression analysis by qRT-PCR. The results showed that, after root treatment with CM-RT, most evaluated genes were upregulated at 24 h after application and downregulated at 48 h after application in roots, while in leaves, genes were downregulated both at 24 and 48 h after application. On the other hand, leaf treatment with CM-RT showed that most evaluated genes in leaves and roots were upregulated at 24 and 48 h after application. These results indicate that the effect of CM-RT applied in roots seems restricted to the applied region and is not sustained, while the application in leaves results in a more systemic response and maintenance of the effect of CM-RT for a longer period. The results obtained in this study emphasize the biotechnological potential of using metabolites of R. tropici as an elicitor of active defense responses in plants.

Overexpression of cotton genes GhDIR4 and GhPRXIIB in Arabidopsis thaliana improves plant resistance to root-knot nematode (Meloidogyne incognita) infection.

Gossypium hirsutum L. represents the best cotton species for fiber production, thus computing the largest cultivated area worldwide. Meloidogyne incognita is a root-knot nematode (RKN) and one of the most important species of Meloidogyne genus, which has a wide host range, including cotton plants. Phytonematode infestations can only be partially controlled by conventional agricultural methods, therefore, more effective strategies to improve cotton resistance to M. incognita disease are highly desirable. The present study employed functional genomics to validate the involvement of two previously identified candidate genes, encoding dirigent protein 4-GhDIR4 and peroxiredoxin-2-GhPRXIIB, in cotton defense against M. incognita. Transgenic A. thaliana plant lines overexpressing GhDIR4 and GhPRXIIB genes were generated and displayed significantly improved resistance against M. incognita infection in terms of female nematode abundance in the roots when compared to wild-type control plants. For our best target-gene GhDIR4, an in-silico functional analysis, including multiple sequence alignment, phylogenetic relationship, and search for specific protein motifs unveiled potential orthologs in other relevant crop plants, including monocots and dicots. Our findings provide valuable information for further understanding the roles of GhDIR and GhPRXIIB genes in cotton defense response against RKN nematode. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03282-4.

Proteome dataset of Hemileia vastatrix by LC-MS/MS label-free identification.

Here we describe the proteome of the fungus Hemileia vastatrix by label free mass spectrometry (LC-MS/MS). H. vastatrix is the causal agent of coffee rust disease, causing great economic losses in this crop. The objective of our work was to identify H. vastatrix proteins potentially involved in host colonization and infection, by exploring the shotgun proteomics approach. A total of 742 proteins were identified and are associated with several crucial molecular functions, biological processes, and cellular components. The proteins identified contribute to a better understanding of the metabolism of the fungus and may help identify target proteins for the development of specific drugs in order to control coffee rust disease. All data can be accessed at the Centre for Computational Mass Spectrometry - MassIVE MSV000087665 -https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=cc71ad75f767451abe72dd1ce0019387.

NBS-LRR-WRKY genes and protease inhibitors (PIs) seem essential for cowpea resistance to root-knot nematode.

Cowpea (Vigna unguiculata L. Walp) is a legume of great economic importance, however it is highly affected by nematodes. The present work aimed to identify proteins and genes involved in nematode resistance by proteomic and transcriptomic analysis. Plants of a genotype resistant (CE31) to root-knot nematode (Meloidogyne spp.) were collected 12 days after inoculation with Meloidogyne incognita and the total proteins and RNA were extracted from the root samples. Shotgun proteomic analysis was performed using an Orbitrap Elite mass spectrometer and the construction and sequencing of cDNA libraries were carried out in a Hi-Seq 2000 sequencing system. The proteomic and transcriptomic analyses revealed key processes involved in cowpea defense and some interesting candidates were further analyzed by RT-qPCR. Proteins and genes involved in essential biological processes were differentially accumulated such as, regulation of transcription, cell wall stiffening and microtubule-based process. However, the main defense strategies of Vigna unguiculata seem to be focused on the interaction of NBS-LRR and WRKY genes for the activation of R genes, production of protease inhibitors and maintenance of actin cytoskeleton. These are key processes that can culminate in the suppression of giant cell formation and consequently in the development of Meloidogyne incognita. SIGNIFICANCE: In this study, we identified proteins and transcripts regulated in cowpea resistant to the nematode Meloidogyne spp. upon inoculation. The results revealed key candidate genes involved in the activation of R genes, the production of protease inhibitors and maintenance of the actin cytoskeleton. These processes might be essential for cowpea resistance, as they can impede nematode nutrition, giant cell formation and consequently the development of Meloidogyne incognita.

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

BACKGROUND: Asian 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. RESULTS: A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (ß/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%. CONCLUSIONS: Our 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.