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.

Overexpression of the GmEXPA1 gene reduces plant susceptibility to Meloidogyne incognita.

KEY MESSAGE: The overexpression of the soybean GmEXPA1 gene reduces plant susceptibility to M. incognita by the increase of root lignification. Plant expansins are enzymes that act in a pH-dependent manner in the plant cell wall loosening and are associated with improved tolerance or resistance to abiotic or biotic stresses. Plant-parasitic nematodes (PPN) can alter the expression profile of several expansin genes in infected root cells. Studies have shown that overexpression or downregulation of particular expansin genes can reduce plant susceptibility to PPNs. Root-knot nematodes (RKN) are obligate sedentary endoparasites of the genus Meloidogyne spp. of which M. incognita is one of the most reported species. Herein, using a transcriptome dataset and real-time PCR assays were identified an expansin A gene (GmEXPA1; Glyma.02G109100) that is upregulated in the soybean nematode-resistant genotype PI595099 compared to the susceptible cultivar BRS133 during plant parasitism by M. incognita. To understand the role of the GmEXPA1 gene during the interaction between soybean plant and M. incognita were generated stable A. thaliana and N. tabacum transgenic lines. Remarkably, both A. thaliana and N. tabacum transgenic lines overexpressing the GmEXPA1 gene showed reduced susceptibility to M. incognita. Furthermore, plant growth, biomass accumulation, and seed yield were not affected in these transgenic lines. Interestingly, significant upregulation of the NtACC oxidase and NtEFE26 genes, involved in ethylene biosynthesis, and NtCCR and Nt4CL genes, involved in lignin biosynthesis, was observed in roots of the N. tabacum transgenic lines, which also showed higher lignin content. These data suggested a possible link between GmEXPA1 gene expression and increased lignification of the root cell wall. Therefore, these data support that engineering of the GmEXPA1 gene in soybean offers a powerful biotechnology tool to assist in RKN management.

Minc03328 effector gene downregulation severely affects Meloidogyne incognita parasitism in transgenic Arabidopsis thaliana.

MAIN CONCLUSION: 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.

Functional characterization of the pUceS8.3 promoter and its potential use for ectopic gene overexpression.

MAIN CONCLUSION: The pUceS8.3 is a constitutive gene promoter with potential for ectopic and strong genes overexpression or active biomolecules in plant tissues attacked by pests, including nematode-induced giant cells or galls. Soybean (Glycine max) is one of the most important agricultural commodities worldwide and a major protein and oil source. Herein, we identified the soybean ubiquitin-conjugating (E2) enzyme gene (GmUBC4; Glyma.18G216000), which is significantly upregulated in response to Anticarsia gemmatalis attack and Meloidogyne incognita-induced galls during plant parasitism by plant nematode. The GmUBC4 promoter sequence and its different modules were functionally characterized in silico and in planta using transgenic Arabidopsis thaliana and G. max lines. Its full-length transcriptional regulatory region (promoter and 5´-UTR sequences, named pUceS8.3 promoter) was able to drive higher levels of uidA (ß-glucuronidase) gene expression in different tissues of transgenic A. thaliana lines compared to its three shortened modules and the p35SdAMV promoter. Notably, higher ß-glucuronidase (GUS) enzymatic activity was shown in M. incognita-induced giant cells when the full pUceS8.3 promoter drove the expression of this reporter gene. Furthermore, nematode-specific dsRNA molecules were successfully overexpressed under the control of the pUceS8.3 promoter in transgenic soybean lines. The RNAi gene construct used here was designed to post-transcriptionally downregulate the previously characterized pre-mRNA splicing factor genes from Heterodera glycines and M. incognita. A total of six transgenic soybean lines containing RNAi gene construct were selected for molecular characterization after infection with M. incognita pre-parasitic second-stage (ppJ2) nematodes. A strong reduction in the egg number produced by M. incognita after parasitism was observed in those transgenic soybean lines, ranging from 71 to 92% compared to wild-type control plants. The present data demonstrated that pUceS8.3 is a gene promoter capable of effectively driving dsRNA overexpression in nematode-induced giant cells of transgenic soybean lines and can be successfully applied as an important biotechnological asset to generate transgenic crops with improved resistance to root-knot nematodes as well as other pests.

Overexpression of a soybean Globin (GmGlb1-1) gene reduces plant susceptibility to Meloidogyne incognita.

MAIN CONCLUSION: The overexpression of the GmGlb1-1 gene reduces plant susceptibility to Meloidogyne incognita. Non-symbiotic globin class #1 (Glb1) genes are expressed in different plant organs, have a high affinity for oxygen, and are related to nitric oxide (NO) turnover. Previous studies showed that soybean Glb1 genes are upregulated in soybean plants under flooding conditions. Herein, the GmGlb1-1 gene was identified in soybean as being upregulated in the nematode-resistant genotype PI595099 compared to the nematode-susceptible cultivar BRS133 during plant parasitism by Meloidogyne incognita. The Arabidopsis thaliana and Nicotiana tabacum transgenic lines overexpressing the GmGlb1-1 gene showed reduced susceptibility to M. incognita. Consistently, gall morphology data indicated that pJ2 nematodes that infected the transgenic lines showed developmental alterations and delayed parasitism progress. Although no significant changes in biomass and seed yield were detected, the transgenic lines showed an elongated, etiolation-like growth under well-irrigation, and also developed more axillary roots under flooding conditions. In addition, transgenic lines showed upregulation of some important genes involved in plant defense response to oxidative stress. In agreement, higher hydrogen peroxide accumulation and reduced activity of reactive oxygen species (ROS) detoxification enzymes were also observed in these transgenic lines. Thus, based on our data and previous studies, it was hypothesized that constitutive overexpression of the GmGlb1-1 gene can interfere in the dynamics of ROS production and NO scavenging, enhancing the acquired systemic acclimation to biotic and abiotic stresses, and improving the cellular homeostasis. Therefore, these collective data suggest that ectopic or nematode-induced overexpression, or enhanced expression of the GmGlb1-1 gene using CRISPR/dCas9 offers great potential for application in commercial soybean cultivars aiming to reduce plant susceptibility to M. incognita.

In planta RNAi approach targeting three M. incognita effector genes disturbed the process of infection and reduced plant susceptibility.

Meloidogyne incognita is the most economically important species of the root-knot nematode complex causing damage to several crops worldwide. During parasitism in host plants, M. incognita secretes several effector proteins to suppress the plant immune system, manipulate the plant cell cycle, and promote parasitism. Several effector proteins have been identified, but their relationship with plant parasitism by M. incognita has not been fully confirmed. Herein, the Minc01696, Minc00344, and Minc00801 putative effector genes were evaluated to assess their importance during soybean and Nicotiana tabacum parasitism by M. incognita. For this study, we used in planta RNAi technology to overexpress dsRNA molecules capable of producing siRNAs that target and downregulate these nematode effector genes. Soybean composite roots and N. tabacum lines were successfully generated, and susceptibility level to M. incognita was evaluated. Consistently, both transgenic soybean roots and transgenic N. tabacum lines carrying the RNAi strategy showed reduced susceptibility to M. incognita. The number of galls per plant and the number of egg masses per plant were reduced by up to 85% in transgenic soybean roots, supported by the downregulation of effector genes in M. incognita during parasitism. Similarly, the number of galls per plant, the number of egg masses per plant, and the nematode reproduction factor were reduced by up to 83% in transgenic N. tabacum lines, which was also supported by the downregulation of the Minc00801 effector gene during parasitism. Therefore, our data indicate that all three effector genes can be a target in the development of new biotechnological tools based on the RNAi strategy in economically important crops for M. incognita control.

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.

Insights obtained using different modules of the cotton uceA1.7 promoter.

MAIN CONCLUSION: The structure of the cotton uceA1.7 promoter and its modules was analyzed; the potential of their key sequences has been confirmed in different tissues, proving to be a good candidate for the development of new biotechnological tools. Transcriptional promoters are among the primary genetic engineering elements used to control genes of interest (GOIs) associated with agronomic traits. Cotton uceA1.7 was previously characterized as a constitutive promoter with activity higher than that of the constitutive promoter from the Cauliflower mosaic virus (CaMV) 35S gene in various plant tissues. In this study, we generated Arabidopsis thaliana homozygous events stably overexpressing the gfp reporter gene driven by different modules of the uceA1.7 promoter. The expression level of the reporter gene in different plant tissues and the transcriptional stability of these modules was determined compared to its full-length promoter and the 35S promoter. The full-length uceA1.7 promoter exhibited higher activity in different plant tissues compared to the 35S promoter. Two modules of the promoter produced a low and unstable transcription level compared to the other promoters. The other two modules rich in cis-regulatory elements showed similar activity levels to full-length uceA1.7 and 35S promoters but were less stable. This result suggests the location of a minimal portion of the promoter that is required to initiate transcription properly (the core promoter). Additionally, the full-length uceA1.7 promoter containing the 5'-untranslated region (UTR) is essential for higher transcriptional stability in various plant tissues. These findings confirm the potential use of the full-length uceA1.7 promoter for the development of new biotechnological tools (NBTs) to achieve higher expression levels of GOIs in, for example, the root or flower bud for the efficient control of phytonematodes and pest-insects, respectively, in important crops.

Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2-ΔΔCt analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 µg g-1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 µg g-1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

Crop losses caused by nematode infections are estimated to be valued at USD 157 billion per year. Meloidogyne incognita, a root-knot nematode (RKN), is considered to be one of the most important plant pathogens due to its worldwide distribution and the austere damage it can cause to a large variety of agronomically important crops. RNA interference (RNAi), a gene silencing process, has proven to be a valuable biotechnology alternative method for RKN control. In this study, the RNAi approach was applied, using fragments of M. incognita genes that encode for two essential molecules, heat-shock protein 90 (HSP90) and isocitrate lyase (ICL). Plant-mediated RNAi of these genes led to a significant level of resistance against M. incognita in the transgenic Nicotiana tabacum plants. Bioassays of plants expressing HSP90 dsRNA demonstrated a delay in gall formation and up to 46% reduction in eggs compared with wild-type plants. A reduction in the level of HSP90 transcripts was observed in recovered eggs from plants expressing dsRNA, indicating that gene silencing persisted and was passed along to first progeny. The ICL knock-down had no clear effect on gall formation but resulted in up to 77% reduction in egg oviposition compared with wild-type plants. Our data suggest that both genes may be involved in RKN development and reproduction. Thus, in this paper, we describe essential candidate genes that could be applied to generate genetically modified crops, using the RNAi strategy to control RKN parasitism.