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Minc03328 effector gene downregulation triggered by in planta RNAi strategy strongly reduced plant susceptibility to Meloidogyne incognita and suggests that Minc03328 gene is a promising target for the development of genetically engineered crops to improve plant tolerance to M. incognita. Meloidogyne incognita is the most economically important species of root-knot nematodes (RKN) and causes severe damage to crops worldwide. M. incognita secretes several effector proteins to suppress the host plant defense response, and manipulate the plant cell cycle and other plant processes facilitating its parasitism. Different secreted effector proteins have already been identified in M. incognita, but not all have been characterized or have had the confirmation of their involvement in nematode parasitism in their host plants. Herein, we characterized the Minc03328 (Minc3s00020g01299) effector gene, confirmed its higher expression in the early stages of M. incognita parasitism in plants, as well as the accumulation of the Minc03328 effector protein in subventral glands and its secretion. We also discuss the potential for simultaneous downregulation of its paralogue Minc3s00083g03984 gene. Using the in planta RNA interference strategy, Arabidopsis thaliana plants overexpressing double-stranded RNA (dsRNA) were generated to specifically targeting and downregulating the Minc03328 gene during nematode parasitism. Transgenic Minc03328-dsRNA lines that significantly downregulated Minc03328 gene expression during M. incognita parasitism were significantly less susceptible. The number of galls, egg masses, and [galls/egg masses] ratio were reduced in these transgenic lines by up to 85%, 90%, and 87%, respectively. Transgenic Minc03328-dsRNA lines showed the presence of fewer and smaller galls, indicating that parasitism was hindered. Overall, data herein strongly suggest that Minc03328 effector protein is important for M. incognita parasitism establishment. As well, the in planta Minc03328-dsRNA strategy demonstrated high biotechnological potential for developing crop species that could efficiently control RKN in the field. Made available in DSpace on 2022-10-03T14:05:21Z (GMT). No. of bitstreams: 1 Minc03328-effector-gene-downregulation-2022.pdf: 6010297 bytes, checksum: 16efd63e19d2622a2a4e30c8810f27cd (MD5) Previous issue date: 2022

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.

This review emphasizes the biotechnological potential of molecules implicated in the different layers of plant immunity, including, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), effector-triggered susceptibility (ETS), and effector-triggered immunity (ETI) that can be applied in the development of disease-resistant genetically modified (GM) plants. These biomolecules are produced by pathogens (viruses, bacteria, fungi, oomycetes) or plants during their mutual interactions. Biomolecules involved in the first layers of plant immunity, PTI and ETS, include inhibitors of pathogen cell-wall-degrading enzymes (CWDEs), plant pattern recognition receptors (PRRs) and susceptibility (S) proteins, while the ETI-related biomolecules include plant resistance (R) proteins. The biomolecules involved in plant defense PTI/ETI responses described herein also include antimicrobial peptides (AMPs), pathogenesis-related (PR) proteins and ribosome-inhibiting proteins (RIPs), as well as enzymes involved in plant defensive secondary metabolite biosynthesis (phytoanticipins and phytoalexins). Moreover, the regulation of immunity by RNA interference (RNAi) in GM disease-resistant plants is also considered. Therefore, the present review does not cover all the classes of biomolecules involved in plant innate immunity that may be applied in the development of disease-resistant GM crops but instead highlights the most common strategies in the literature, as well as their advantages and disadvantages.

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter.