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 dCas9 HAT were first generated. Then, we showed that the CRISPRa dCas9 HAT mechanism increased the promoter activity controlling the β-glucuronidase ( GUS ) reporter gene. To activate the endogenous promoter of AREB1 , the CRISPRa dCas9 HAT 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 dCas9 HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.

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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Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil (Anthonomus grandis), we showed that the chimeric protein PTD-DRBD (peptide transduction domain—dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.

The initial greening of angiosperm occurs upon light-activation of photoreceptors that trigger photomorphogenesis followed with the development of chloroplasts. In these semiautonomous organelles, the construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here we show that PAP8, as an essential subunit of the plastid-encoded RNA polymerase, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 is localized and active in both plastids and the nucleus and particularly essential for the formation of late photobodies. In the albino pap8 mutant, phytochrome-mediated signalling is altered, PIFs are maintained, HY5 is not stabilized, and GLK1 expression is impaired. PAP8 translocates into plastids losing its pre-sequence, interacts with the PEP, and using an unknown route or a retrograde transport, reaches the nucleus where it has the ability to interact with pTAC12/HMR/PAP5. Since PAP8 is required for the phytochrome-B-mediated signalling cascade and the reshaping of the PEP, it may coordinate nuclear gene expression with the PEP-driven chloroplastic gene expression during chloroplast biogenesis.

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.

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.

The initial greening of angiosperm occurs upon light-activation of photoreceptors that trigger photomorphogenesis followed with the development of chloroplasts. In these semiautonomous organelles, the construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here we show that PAP8, as an essential subunit of the plastid-encoded RNA polymerase, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 is localized and active in both plastids and the nucleus and particularly essential for the formation of late photobodies.In the albino pap8 mutant, phytochrome-mediated signalling is altered, PIFs are maintained, HY5 is not stabilized, and GLK1 expression is impaired. PAP8 translocates into plastids losing its pre-sequence, interacts with the PEP, and using an unknown route or a retrograde transport, reaches the nucleus where it has the ability to interact with pTAC12/HMR/PAP5.