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.

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.

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.

MALDI TOF MS-profiling: Applications for bacterial and plant sample differentiation and biological variability assessment.

In this study, we evaluated the potential use of MALDI-TOF MS Profiling for the differentiation of biological samples submitted to different treatments. We compared the bacterium Xanthomonas campestris pv. campestris (Xcc), grown in culture medium and in vivo (recovered from the plant). Plant samples were also analyzed and included explants at different somatic embryogenesis (SE) stages, as well as leaves from Brassica oleracea and Arabidopsis thaliana inoculated with Xcc, at different time points. The results showed that bacteria and highly divergent plant samples, such as those from embryogenic stages, can be unequivocally differentiated and the clustering was in accordance with proteomic analysis performed by 2-DE. These results show an important application of MALDI-TOF MS Profiling to select and prioritize samples to be analyzed prior to more complex approaches including transcriptomics and proteomics. We also show that in plant-pathogen interactions, when more subtle differences are obtained, the main contribution of MALDI-TOF MS Profiling is in the assessment of experimental variability. This is relevant since reproducibility is a challenging issue when dealing with complex experimental conditions such as plant-pathogen interactions. We propose the use of MALDI-TOF MS Profiling to aid researchers in minimizing experimental variability unrelated to the condition being analyzed. SIGNIFICANCE: MALDI-Profiling offers an inexpensive, rapid and reliable approach for investigating the protein profile to assess sample differentiation and experimental variability in microorganisms and plants and can be highly useful to analyze samples prior to more complex and expensive techniques such as proteomics and transcriptomics.

Genotype-dependent changes of gene expression during somatic embryogenesis in oil palm hybrids (Elaeis oleifera x E. guineensis).

To understand the molecular processes triggered during the different steps of somatic embryogenesis (SE) in oil palm, the expression of 19 genes associated to SE identified in proteomic and transcriptomic studies was investigated by qRT-PCR. To evaluate the differential expression of these genes, two interspecific hybrid genotypes (Elaeis oleifera x Elaeis guineensis) contrasting for the acquisition of embryogenic competence were used. Aclorophyllated leaves of both hybrids, one responsive (B351733) and the other non-responsive (B352933) to SE were submitted to callus induction and collected at different time points: 0 (before induction), 14, 30, 90 and 150 days of callus induction (doi). The results obtained showed that all evaluated genes were downregulated at 14 doi in the responsive genotype when compared to the non-responsive. It was also possible to observe that most of the genes changed their expression behavior at 30 doi and were upregulated thereafter until 150 doi, with the exception of the pathogenesis-related PRB1-3-like (PRB1-3) gene, which did not show differential expression at 30 doi and was downregulated at 90 and 150 doi when compared to the non-responsive hybrid. These results indicate that 30 doi is a turning point in gene expression, probably associated to embryogenic competence acquisition. We also show that the expression behavior of the responsive genotype is more stable than that of the non-responsive when the different induction time points are compared to 0 doi (before induction). Moreover, the results obtained in this study corroborate our hypothesis that the regulation of genes involved in the control of oxidative stress and energy metabolism are crucial for the acquisition of embryogenic competence in oil palm.