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Meloidogyne incognita is a plant-parasitic root-knot nematode (RKN, PPN) responsible for causing damage to several crops worldwide. In Caenorhabditis elegans, the DAF-16 and SKN-1 transcription factors (TFs) orchestrate aging, longevity, and defense responses to several stresses. Here, we report that MiDaf16-like1 and MiSkn1-like1, which are orthologous to DAF-16 and SKN-1 in C. elegans, and some of their targets, are modulated in M. incognita J2 during oxidative stress or plant parasitism. We used RNAi technology for the stable production of siRNAs in planta to downregulate the MiDaf16-like1 and MiSkn1-like1 genes of M. incognita during host plant parasitism. Arabidopsis thaliana and Nicotiana tabacum overexpressing a hairpin-derived dsRNA targeting these genes individually (single-gene silencing) or simultaneously (double-gene silencing) were generated. T2 plants were challenged with M. incognita and the number of eggs, galls, and J2, and the nematode reproduction factor (NRF) were evaluated. Our data indicate that MiDaf16-like1, MiSkn1-like1 and some genes from their networks are modulated in M. incognita J2 during oxidative stress or plant parasitism. Transgenic A. thaliana and N. tabacum plants with single- or double-gene silencing showed significant reductions in the numbers of eggs, J2, and galls, and in NRF. Additionally, the double-gene silencing plants had the highest resistance level. Gene expression assays confirmed the downregulation of the MiDaf16-like1 and MiSkn1-like1 TFs and defense genes in their networks during nematode parasitism in the transgenic plants. All these findings demonstrate that these two TFs are potential targets for the development of biotechnological tools for nematode control and management in economically important crops.

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Drought episodes decrease plant growth and productivity, which in turn cause high economic losses. Plants naturally sense and respond to water stress by activating specific signalling pathways leading to physiological and developmental adaptations. Genetically engineering genes that belong to these pathways might improve the drought tolerance of plants. The abscisic acid (ABA)-responsive element binding protein 1/ABRE binding factor (AREB1/ABF2) is a key positive regulator of the drought stress response. We investigated whether the CRISPR activation (CRISPRa) system that targets AREB1 might contribute to improve drought stress tolerance in Arabidopsis. Arabidopsis histone acetyltransferase 1 (AtHAT1) promotes gene expression activation by switching chromatin to a relaxed state. Stable transgenic plants expressing chimeric dCas9HAT were first generated. Then, we showed that the CRISPRa dCas9HAT mechanism increased the promoter activity controlling the ß-glucuronidase (GUS) reporter gene. To activate the endogenous promoter of AREB1, the CRISPRa dCas9HAT system was set up, and resultant plants showed a dwarf phenotype. Our qRT-PCR experiments indicated that both AREB1 and RD29A, a gene positively regulated by AREB1, exhibited higher gene expression than the control plants. The plants generated here showed higher chlorophyll content and faster stomatal aperture under water deficit, in addition to a better survival rate after drought stress. Altogether, we report that CRISPRa dCas9HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.

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The pathogenicity of phytonematodes relies on secreted virulence factors to rewire host cellular pathways for the benefits of the nematode. In the root-knot nematode (RKN) Meloidogyne incognita, thousands of predicted secreted proteins have been identified and are expected to interact with host proteins at different developmental stages of the parasite. Identifying the host targets will provide compelling evidence about the biological significance and molecular function of the predicted proteins. Here, we have focused on the hub protein CSN5, the fifth subunit of the pleiotropic and eukaryotic conserved COP9 signalosome (CSN), which is a regulatory component of the ubiquitin/proteasome system. We used affinity purification-mass spectrometry (AP-MS) to generate the interaction network of CSN5 in M. incognita-infected roots. We identified the complete CSN complex and other known CSN5 interaction partners in addition to unknown plant and M. incognita proteins. Among these, we described M. incognita PASSE-MURAILLE (MiPM), a small pioneer protein predicted to contain a secretory peptide that is up-regulated mostly in the J2 parasitic stage. We confirmed the CSN5-MiPM interaction, which occurs in the nucleus, by bimolecular fluorescence complementation (BiFC). Using MiPM as bait, a GST pull-down assay coupled with MS revealed some common protein partners between CSN5 and MiPM. We further showed by in silico and microscopic analyses that the recombinant purified MiPM protein enters the cells of Arabidopsis root tips in a non-infectious context. In further detail, the supercharged N-terminal tail of MiPM (NTT-MiPM) triggers an unknown host endocytosis pathway to penetrate the cell. The functional meaning of the CSN5-MiPM interaction in the M. incognita parasitism is discussed. Moreover, we propose that the cell-penetrating properties of some M. incognita secreted proteins might be a non-negligible mechanism for cell uptake, especially during the steps preceding the sedentary parasitic phase.

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Tripartite interactions between legumes and their root symbionts (rhizobia and arbuscular mycorrhizal fungi, AMF) are poorly understood, although it is well established that only specific combinations of symbionts lead to optimal plant growth. A classic example in which to investigate such interactions is the Brazilian legume tree Piptadenia gonoacantha (Caesalpinioideae), for which efficient nodulation has been described as dependent on the presence of AMF symbiosis. In this study, we compared the nodulation behaviour of several rhizobial strains with or without AMF inoculation, and performed analyses on nodulation, nodule cytology, N-fixing efficiency, and plant growth response. Nodulation of P. gonoacantha does not rely on the presence of AMF, but mycorrhization was rhizobial strain-dependent, and nodule effectiveness and plant growth were dependent on the presence of specific combinations of rhizobial strains and AMF. The co-occurrence of both symbionts within efficient nodules and the differentiation of bacteroids within nodule cells were also demonstrated. Novel close interactions and interdependency for the establishment and/or functioning of these symbioses were also revealed in Piptadenia, thanks to immunocytochemical analyses. These data are discussed in terms of the evolutionary position of the newly circumscribed mimosoid clade within the Caesalpinioid subfamily and its relative proximity to non-nodulated (but AMF-associated) basal subfamilies.

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Some species of the genus Paraburkholderia that are able to nodulate and fix nitrogen in symbiosis with legumes are called ß-rhizobia and represent a group of ecological and biotechnological importance. We used Mimosa pudica and Phaseolus vulgaris to trap 427 rhizobial isolates from rhizospheric soil of Mimoseae trees in the Brazilian Atlantic Forest. Eighty-four representative strains were selected according to the 16S rRNA haplotypes and taxonomically characterized using a concatenated 16S rRNA-recA phylogeny. Most strains were assembled in the genus Paraburkholderia, including Paraburkholderia sabiae and Pa. nodosa. Mesorhizobium (α-rhizobia) and Cupriavidus (ß-rhizobia) were also isolated, but in smaller proportions. Multilocus sequence analysis and BOX-PCR analyses indicated that six clusters of Paraburkholderia represent potential new species. In the phylogenetic analysis of the nodC gene, the majority of the strains were positioned in the same groups as in the 16S rRNA-recA tree, indicative of stability and vertical inheritance, but we also identified horizontal transfer of nodC in Pa. sabiae. All α- and ß-rhizobial species were trapped by both legumes, although preferences of the host plants for specific rhizobial species have been observed.

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BACKGROUND: Plant-parasitic nematode interactions occur within a vast molecular plant immunity network. Following initial contact with the host plant roots, plant-parasitic nematodes (PPNs) activate basal immune responses. Defence priming involves the release in the apoplast of toxic molecules derived from reactive species or secondary metabolism. In turn, PPNs must overcome the poisonous and stressful environment at the plant-nematode interface. The ability of PPNs to escape this first line of plant immunity is crucial and will determine its virulence. SCOPE: Nematodes trigger crucial regulatory cytoprotective mechanisms, including antioxidant and detoxification pathways. Knowledge of the upstream regulatory components that contribute to both of these pathways in PPNs remains elusive. In this review, we discuss how PPNs probably orchestrate cytoprotection to resist plant immune responses, postulating that it may be derived from ancient molecular mechanisms. The review focuses on two transcription factors, DAF-16 and SKN-1 , which are conserved in the animal kingdom and are central regulators of cell homeostasis and immune function. Both regulate the unfolding protein response and the antioxidant and detoxification pathways. DAF-16 and SKN-1 target a broad spectrum of Caenorhabditis elegans genes coding for numerous protein families present in the secretome of PPNs. Moreover, some regulatory elements of DAF-16 and SKN-1 from C. elegans have already been identified as important genes for PPN infection. CONCLUSION: DAF-16 and SKN-1 genes may play a pivotal role in PPNs during parasitism. In the context of their hub status and mode of regulation, we suggest alternative strategies for control of PPNs through RNAi approaches.

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During a survey of root-nodulating symbionts of Mimosoid species in the south-east region of Brazil, eight Paraburkholderia isolates were obtained from nodules of the legume species Piptadenia gonoacantha, either from the field or following a soil trapping method with the same plant host. 16S rRNA gene as well as recA and gyrB phylogenetic markers placed these strains in two new clades within the genus Burkholderia sensu lato. DNA-DNA hybridization values and analyses of average nucleotide identities of the whole genome sequence of selected strains in each clade (STM 7183 and STM 7296) showed that the two clades represented novel species of the genus Paraburkholderia. All eight isolates were further characterized using DNA base content determination, chemotaxonomic and biochemical profiling and symbiotic properties, which allowed to distinguish the novel species from known diazotrophic species of the genus Paraburkholderia. Based on genomic and phenotypic data, the names Paraburkholderia piptadeniae sp. nov. with type strain STM 7183T (=DSM 101189T=LMG 29163T) and Paraburkholderia ribeironis sp. nov. with type strain STM 7296T (=DSM 101188T=LMG 29351T) are proposed.

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Burkholderia legume symbionts (also called α-rhizobia) are ancient in origin and are the main nitrogen-fixing symbionts of species belonging to the large genus Mimosa in Brazil. We investigated the extent of the affinity between Burkholderia and species in the tribe Mimoseae by studying symbionts of the genera Piptadenia (P.), Parapiptadenia (Pp.), Pseudopiptadenia (Ps.), Pityrocarpa (Py.), Anadenanthera (A.) and Microlobius (Mi.), all of which are native to Brazil and are phylogenetically close to Mimosa, and which together with Mimosa comprise the "Piptadenia group". We characterized 196 strains sampled from 18 species from 17 locations in Brazil using two neutral markers and two symbiotic genes in order to assess their species affiliations and the evolution of their symbiosis genes. We found that Burkholderia are common and highly diversified symbionts of species in the Piptadenia group, comprising nine Burkholderia species, of which three are new ones and one was never reported as symbiotic (B. phenoliruptrix). However, α-rhizobia were also detected and were occasionally dominant on a few species. A strong sampling site effect on the rhizobial nature of symbionts was detected, with the symbiont pattern of the same legume species changing drastically from location to location, even switching from ß to α-rhizobia. Coinoculation assays showed a strong affinity of all the Piptadenia group species towards Burkholderia genotypes, with the exception of Mi. foetidus. Phylogenetic analyses of neutral and symbiotic markers showed that symbiosis genes in Burkholderia from the Piptadenia group have evolved mainly through vertical transfer, but also by horizontal transfer in two species.

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