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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.

The Coffea arabica HB12 gene (CaHB12), which encodes a transcription factor belonging to the HD-Zip I subfamily, is upregulated under drought, and its constitutive overexpression (35S:CaHB12OX) improves the Arabidopsis thaliana tolerance to drought and salinity stresses. Herein, we generated transgenic cotton events constitutively overexpressing the CaHB12 gene, characterized these events based on their increased tolerance to water deficit, and exploited the gene expression level from the CaHB12 network. The segregating events Ev8.29.1, Ev8.90.1, and Ev23.36.1 showed higher photosynthetic yield and higher water use efficiency under severe water deficit and permanent wilting point conditions compared to wild-type plants. Under well-irrigated conditions, these three promising transformed events showed an equivalent level of Abscisic acid (ABA) and decreased Indole-3-acetic acid (IAA) accumulation, and a higher putrescine/(spermidine + spermine) ratio in leaf tissues was found in the progenies of at least two transgenic cotton events compared to non-transgenic plants. In addition, genes that are considered as modulated in the A. thaliana 35S:CaHB12OX line were also shown to be modulated in several transgenic cotton events maintained under field capacity conditions. The upregulation of GhPP2C and GhSnRK2 in transgenic cotton events maintained under permanent wilting point conditions suggested that CaHB12 might act enhancing the ABA-dependent pathway. All these data confirmed that CaHB12 overexpression improved the tolerance to water deficit, and the transcriptional modulation of genes related to the ABA signaling pathway or downstream genes might enhance the defense responses to drought. The observed decrease in IAA levels indicates that CaHB12 overexpression can prevent leaf abscission in plants under or after stress. Thus, our findings provide new insights on CaHB12 gene and identify several promising cotton events for conducting field trials on water deficit tolerance and agronomic performance.