The regeneration conferring transcription factor complex ERF115-PAT1 coordinates a wound-induced response in root-knot nematode induced galls.

The establishment of root-knot nematode (RKN; Meloidogyne spp.) induced galls in the plant host roots likely involves a wound-induced regeneration response. Confocal imaging demonstrates physical stress or injury caused by RKN infection during parasitism in the model host Arabidopsis thaliana. The ERF115-PAT1 heterodimeric transcription factor complex plays a recognized role in wound-induced regeneration. ERF115 and PAT1 expression flanks injured gall cells likely driving mechanisms of wound healing, implying a local reactivation of cell division which is also hypothetically involved in gall genesis. Herein, functional investigation revealed that ectopic ERF115 expression resulted in premature induction of galls, and callus formation adjacent to the expanding female RKN was seen upon PAT1 upregulation. Smaller galls and less reproduction were observed in ERF115 and PAT1 knockouts. Investigation of components in the ERF115 network upon overexpression and knockdown by qRT-PCR suggests it contributes to steer gall wound-sensing and subsequent competence for tissue regeneration. High expression of CYCD6;1 was detected in galls, and WIND1 overexpression resulted in similar ERF115OE gall phenotypes, also showing faster gall induction. Along these lines, we show that the ERF115-PAT1 complex likely coordinates stress signalling with tissue healing, keeping the gall functional until maturation and nematode reproduction.

A novel soybean hairy root system for gene functional validation.

Agrobacterium rhizogenes-mediated transformation has long been explored as a versatile and reliable method for gene function validation in many plant species, including soybean (Glycine max). Likewise, detached-leaf assays have been widely used for rapid and mass screening of soybean genotypes for disease resistance. The present study combines these two methods to establish an efficient and practical system to generate transgenic soybean hairy roots from detached leaves and their subsequent culture under ex vitro conditions. We demonstrated that hairy roots derived from leaves of two (tropical and temperate) soybean cultivars could be successfully infected by economically important species of root-knot nematodes (Meloidogyne incognita and M. javanica). The established detached-leaf method was further explored for functional validation of two candidate genes encoding for cell wall modifying proteins (CWMPs) to promote resistance against M. incognita through distinct biotechnological strategies: the overexpression of a wild Arachis α-expansin transgene (AdEXPA24) and the dsRNA-mediated silencing of an endogenous soybean polygalacturonase gene (GmPG). AdEXPA24 overexpression in hairy roots of RKN-susceptible soybean cultivar significantly reduced nematode infection by approximately 47%, whereas GmPG downregulation caused an average decrease of 37%. This novel system of hairy root induction from detached leaves showed to be an efficient, practical, fast, and low-cost method suitable for high throughput in root analysis of candidate genes in soybean.

Pyramiding dsRNAs increases phytonematode tolerance in cotton plants.

MAIN CONCLUSION: Host-derived suppression of nematode essential genes decreases reproduction of Meloidogyne incognita in cotton. Root-knot nematodes (RKN) represent one of the most damaging plant-parasitic nematode genera worldwide. RNAi-mediated suppression of essential nematode genes provides a novel biotechnological strategy for the development of sustainable pest-control methods. Here, we used a Host Induced Gene Silencing (HIGS) approach by stacking dsRNA sequences into a T-DNA construct to target three essential RKN genes: cysteine protease (Mi-cpl), isocitrate lyase (Mi-icl), and splicing factor (Mi-sf), called dsMinc1, driven by the pUceS8.3 constitutive soybean promoter. Transgenic dsMinc1-T4 plants infected with Meloidogyne incognita showed a significant reduction in gall formation (57-64%) and egg masses production (58-67%), as well as in the estimated reproduction factor (60-78%), compared with the susceptible non-transgenic cultivar. Galls of the RNAi lines are smaller than the wild-type (WT) plants, whose root systems exhibited multiple well-developed root swellings. Transcript levels of the three RKN-targeted genes decreased 13- to 40-fold in nematodes from transgenic cotton galls, compared with those from control WT galls. Finally, the development of non-feeding males in transgenic plants was 2-6 times higher than in WT plants, indicating a stressful environment for nematode development after RKN gene silencing. Data strongly support that HIGS of essential RKN genes is an effective strategy to improve cotton plant tolerance. This study presents the first application of dsRNA sequences to target multiple genes to promote M. incognita tolerance in cotton without phenotypic penalty in transgenic plants.

Senescence-Associated Glycine max (Gm)NAC Genes: Integration of Natural and Stress-Induced Leaf Senescence.

Leaf senescence is a genetically regulated developmental process that can be triggered by a variety of internal and external signals, including hormones and environmental stimuli. Among the senescence-associated genes controlling leaf senescence, the transcriptional factors (TFs) comprise a functional class that is highly active at the onset and during the progression of leaf senescence. The plant-specific NAC (NAM, ATAF, and CUC) TFs are essential for controlling leaf senescence. Several members of Arabidopsis AtNAC-SAGs are well characterized as players in elucidated regulatory networks. However, only a few soybean members of this class display well-known functions; knowledge about their regulatory circuits is still rudimentary. Here, we describe the expression profile of soybean GmNAC-SAGs upregulated by natural senescence and their functional correlation with putative AtNAC-SAGs orthologs. The mechanisms and the regulatory gene networks underlying GmNAC081- and GmNAC030-positive regulation in leaf senescence are discussed. Furthermore, new insights into the role of GmNAC065 as a negative senescence regulator are presented, demonstrating extraordinary functional conservation with the Arabidopsis counterpart. Finally, we describe a regulatory circuit which integrates a stress-induced cell death program with developmental leaf senescence via the NRP-NAC-VPE signaling module.

Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods.

MAIN CONCLUSION: The combined Agrobacterium- and biolistic-mediated methods of cotton transformation provide a straightforward and highly efficient protocol for obtaining transgenic cotton. Cotton (Gossypium spp.) is the most important crop for natural textile fiber production worldwide. Nonetheless, one of the main challenges in cotton production are the losses resulting from insect pests, pathogens, and abiotic stresses. One effective way to solve these issues is to use genetically modified (GM) varieties. Herein, we describe an improved protocol for straightforward and cost-effective genetic transformation of cotton embryo axes, merging biolistics and Agrobacterium. The experimental steps include (1) Agrobacterium preparation, (2) seed sterilization, (3) cotton embryo excision, (4) lesion of shoot-cells by tungsten bombardment, (5) Agrobacterium-mediated transformation, (6) embryo co-culture, (7) regeneration and selection of transgenic plants in vitro, and (8) molecular characterization of plants. Due to the high regenerative power of the embryonic axis and the exceptional ability of the meristem cells for plant regeneration through organogenesis in vitro, this protocol can be performed in approximately 4-10 weeks, with an average plant regeneration of about 5.5% (± 0.53) and final average transformation efficiency of 60% (± 0.55). The transgene was stably inherited, and most transgenic plants hold a single copy of the transgene, as desirable and expected in Agrobacterium-mediated transformation. Additionally, the transgene was stably expressed over generations, and transgenic proteins could be detected at high levels in the T2 generation of GM cotton plants. The T2 progeny showed no phenotypic or productivity disparity compared to wild-type plants. Collectively, the use of cotton embryo axes and the enhanced DNA-delivery system by combining particle bombardment and Agrobacterium infection enabled efficient transgenic plant recovery, overcoming usual limitations associated with the recalcitrance of several cotton genotypes subjected to somatic embryogenesis. The improved approach states this method's success for cotton genetic modification, allowing us to obtain GM cotton plants carrying traits, which are of fundamental relevance for the advancement of global agribusiness.

The Armadillo BTB Protein ABAP1 Is a Crucial Player in DNA Replication and Transcription of Nematode-Induced Galls.

The biogenesis of root-knot nematode (Meloidogyne spp.)-induced galls requires the hyperactivation of the cell cycle with controlled balance of mitotic and endocycle programs to keep its homeostasis. To better understand gall functioning and to develop new control strategies for this pest, it is essential to find out how the plant host cell cycle programs are responding and integrated during the nematode-induced gall formation. This work investigated the spatial localization of a number of gene transcripts involved in the pre-replication complex during DNA replication in galls and report their akin colocation with the cell cycle S-phase regulator Armadillo BTB Arabidopsis Protein 1 (ABAP1). ABAP1 is a negative regulator of pre-replication complex controlling DNA replication of genes involved in control of cell division and proliferation; therefore, its function has been investigated during gall ontogenesis. Functional analysis was performed upon ABAP1 knockdown and overexpression in Arabidopsis thaliana. We detected ABAP1 promoter activity and localized ABAP1 protein in galls during development, and its overexpression displayed significantly reduced gall sizes containing atypical giant cells. Profuse ABAP1 expression also impaired gall induction and hindered nematode reproduction. Remarkably, ABAP1 knockdown likewise negatively affected gall and nematode development, suggesting its involvement in the feeding site homeostasis. Microscopy analysis of cleared and nuclei-stained whole galls revealed that ABAP1 accumulation resulted in aberrant giant cells displaying interconnected nuclei filled with enlarged heterochromatic regions. Also, imbalanced ABAP1 expression caused changes in expression patterns of genes involved in the cell division control as demonstrated by qRT-PCR. CDT1a, CDT1b, CDKA;1, and CYCB1;1 mRNA levels were significantly increased in galls upon ABAP1 overexpression, possibly contributing to the structural changes in galls during nematode infection. Overall, data obtained in galls reinforced the role of ABAP1 controlling DNA replication and mitosis and, consequently, cell proliferation. ABAP1 expression might likely take part of a highly ordered mechanism balancing of cell cycle control to prevent gall expansion. ABAP1 expression might prevent galls to further expand, limiting excessive mitotic activity. Our data strongly suggest that ABAP1 as a unique plant gene is an essential component for cell cycle regulation throughout gall development during nematode infection and is required for feeding site homeostasis.

Contrasting roles of GmNAC065 and GmNAC085 in natural senescence, plant development, multiple stresses and cell death responses.

NACs are plant-specific transcription factors involved in controlling plant development, stress responses, and senescence. As senescence-associated genes (SAGs), NACs integrate age- and stress-dependent pathways that converge to programmed cell death (PCD). In Arabidopsis, NAC-SAGs belong to well-characterized regulatory networks, poorly understood in soybean. Here, we interrogated the soybean genome and provided a comprehensive analysis of senescence-associated Glycine max (Gm) NACs. To functionally examine GmNAC-SAGs, we selected GmNAC065, a putative ortholog of Arabidopsis ANAC083/VNI2 SAG, and the cell death-promoting GmNAC085, an ANAC072 SAG putative ortholog, for analyses. Expression analysis of GmNAC065 and GmNAC085 in soybean demonstrated (i) these cell death-promoting GmNACs display contrasting expression changes during age- and stress-induced senescence; (ii) they are co-expressed with functionally different gene sets involved in stress and PCD, and (iii) are differentially induced by PCD inducers. Furthermore, we demonstrated GmNAC065 expression delays senescence in Arabidopsis, a phenotype associated with enhanced oxidative performance under multiple stresses, higher chlorophyll, carotenoid and sugar contents, and lower stress-induced PCD compared to wild-type. In contrast, GmNAC085 accelerated stress-induced senescence, causing enhanced chlorophyll loss, ROS accumulation and cell death, decreased antioxidative system expression and activity. Accordingly, GmNAC065 and GmNAC085 targeted functionally contrasting sets of downstream AtSAGs, further indicating that GmNAC85 and GmNAC065 regulators function inversely in developmental and environmental PCD.

Dissecting protein domain variability in the core RNA interference machinery of five insect orders.

RNA interference (RNAi)-mediated gene silencing can be used to control specific insect pest populations. Unfortunately, the variable efficiency in the knockdown levels of target genes has narrowed the applicability of this technology to a few species. Here, we examine the current state of knowledge regarding the miRNA (micro RNA) and siRNA (small interfering RNA) pathways in insects and investigate the structural variability at key protein domains of the RNAi machinery. Our goal was to correlate domain variability with mechanisms affecting the gene silencing efficiency. To this end, the protein domains of 168 insect species, encompassing the orders Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, were analysed using our pipeline, which takes advantage of meticulous structure-based sequence alignments. We used phylogenetic inference and the evolutionary rate coefficient (K) to outline the variability across domain regions and surfaces. Our results show that four domains, namely dsrm, Helicase, PAZ and Ribonuclease III, are the main contributors of protein variability in the RNAi machinery across different insect orders. We discuss the potential roles of these domains in regulating RNAi-mediated gene silencing and the role of loop regions in fine-tuning RNAi efficiency. Additionally, we identified several order-specific singularities which indicate that lepidopterans have evolved differently from other insect orders, possibly due to constant coevolution with plants and viruses. In conclusion, our results highlight several variability hotspots that deserve further investigation in order to improve the application of RNAi technology in the control of insect pests.

Transcriptional modulation of AREB-1 by CRISPRa improves plant physiological performance under severe water deficit.

Plants are sessile organisms, which are vulnerable to environmental stresses. As such, plants have developed multiple molecular, physiological, and cellular mechanisms to cope with natural stressors. However, these environmental adversities, including drought, are sources of the main agribusiness problems since they interfere with plant growth and productivity. Particularly under water deprivation conditions, the abscisic acid-responsive element-binding protein AREB1/ABF2 plays an important role in drought stress response and physiological adaptation. In this investigation, we provide substantial confirmation for the role of AREB1/ABF2 in plant survival under severe water deficit using the CRISPR activation (CRISPRa) technique to enhance the AREB1 gene expression. In our strategy, the inactive nuclease dCas9 was fused with an Arabidopsis histone acetyltransferase 1, which improves gene expression by remodeling chromatin. The AREB1 overexpression promotes an improvement in the physiological performance of the transgenic homozygous plants under drought, which was associated with an increase in chlorophyll content, antioxidant enzyme activity, and soluble sugar accumulation, leading to lower reactive oxygen species accumulation. Finally, we found that the CRISPR-mediated up-regulation of AREB1 changes the abundance of several downstream ABA-inducible genes, allowing us to report that CRISPRa dCas9-HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.

Evolutionarily conserved plant genes responsive to root-knot nematodes identified by comparative genomics.

Root-knot nematodes (RKNs, genus Meloidogyne) affect a large number of crops causing severe yield losses worldwide, more specifically in tropical and sub-tropical regions. Several plant species display high resistance levels to Meloidogyne, but a general view of the plant immune molecular responses underlying resistance to RKNs is still lacking. Combining comparative genomics with differential gene expression analysis may allow the identification of widely conserved plant genes involved in RKN resistance. To identify genes that are evolutionary conserved across plant species, we used OrthoFinder to compared the predicted proteome of 22 plant species, including important crops, spanning 214 Myr of plant evolution. Overall, we identified 35,238 protein orthogroups, of which 6,132 were evolutionarily conserved and universal to all the 22 plant species (PLAnts Common Orthogroups-PLACO). To identify host genes responsive to RKN infection, we analyzed the RNA-seq transcriptome data from RKN-resistant genotypes of a peanut wild relative (Arachis stenosperma), coffee (Coffea arabica L.), soybean (Glycine max L.), and African rice (Oryza glaberrima Steud.) challenged by Meloidogyne spp. using EdgeR and DESeq tools, and we found 2,597 (O. glaberrima), 743 (C. arabica), 665 (A. stenosperma), and 653 (G. max) differentially expressed genes (DEGs) during the resistance response to the nematode. DEGs' classification into the previously characterized 35,238 protein orthogroups allowed identifying 17 orthogroups containing at least one DEG of each resistant Arachis, coffee, soybean, and rice genotype analyzed. Orthogroups contain 364 DEGs related to signaling, secondary metabolite production, cell wall-related functions, peptide transport, transcription regulation, and plant defense, thus revealing evolutionarily conserved RKN-responsive genes. Interestingly, the 17 DEGs-containing orthogroups (belonging to the PLACO) were also universal to the 22 plant species studied, suggesting that these core genes may be involved in ancestrally conserved immune responses triggered by RKN infection. The comparative genomic approach that we used here represents a promising predictive tool for the identification of other core plant defense-related genes of broad interest that are involved in different plant-pathogen interactions.

Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a Histone AcetylTransferase.

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.

The Stress-Induced Soybean NAC Transcription Factor GmNAC81 Plays a Positive Role in Developmentally Programmed Leaf Senescence.

The onset of leaf senescence is a highly regulated developmental change that is controlled by both genetics and the environment. Senescence is triggered by massive transcriptional reprogramming, but functional information about its underlying regulatory mechanisms is limited. In the current investigation, we performed a functional analysis of the soybean (Glycine max) osmotic stress- and endoplasmic reticulum (ER) stress-induced NAC transcription factor GmNAC81 during natural leaf senescence using overexpression studies and reverse genetics. GmNAC81-overexpressing lines displayed accelerated flowering and leaf senescence but otherwise developed normally. The precocious leaf senescence of GmNAC81-overexpressing lines was associated with greater Chl loss, faster photosynthetic decay and higher expression of hydrolytic enzyme-encoding GmNAC81 target genes, including the vacuolar processing enzyme (VPE), an executioner of vacuole-triggered programmed cell death (PCD). Conversely, virus-induced gene silencing-mediated silencing of GmNAC81 delayed leaf senescence and was associated with reductions in Chl loss, lipid peroxidation and the expression of GmNAC81 direct targets. Promoter-reporter studies revealed that the expression pattern of GmNAC81 was associated with senescence in soybean leaves. Our data indicate that GmNAC81 is a positive regulator of age-dependent senescence and may integrate osmotic stress- and ER stress-induced PCD responses with natural leaf senescence through the GmNAC81/VPE regulatory circuit.