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.

Discovery and functional characterization of novel cotton promoters with potential application to pest control.

KEY MESSAGE: pGhERF105 and pGhNc-HARBI1 promoters are highly responsive to CBW infestation and exhibit strong activity in vegetative and reproductive tissues, increasing their potential application in GM crop plants for pest control. The main challenge to cotton (Gossypium hirsutum) crop productivity is the constant attack of several pests, including the cotton boll weevil (CBW, Anthonomus grandis), which uses cotton floral buds for feeding and egg-laying. The endophytic nature of the early developmental stages of CBW makes conventional pesticide-based control poorly efficient. Most biotechnological assets used for pest control are based on Bacillus thurigiensis insecticidal Cry toxins or the silencing of insect-pest essential genes using RNA-interference technology. However, suitable plant promoter sequences are required to efficiently drive insecticidal molecules to the target plant tissue. This study selected the Ethylene Responsive Factor 105 (GhERF105) and Harbinger transposase-derived nuclease (GhNc-HARBI1) genes based on available transcriptome-wide data from cotton plants infested by CBW larvae. The GhERF105 and GhNc-HARBI1 genes showed induction kinetics from 2 to 96 h under CBW's infestation in cotton floral buds, uncovering the potential application of their promoters. Therefore, the promoter regions (1,500 base pairs) were assessed and characterized using Arabidopsis thaliana transgenic plants. The pGhERF105 and pGhNc-HARBI1 promoters showed strong activity in plant vegetative (leaves and roots) and reproductive (flowers and fruits) tissues, encompassing higher GUS transcriptional activity than the viral-constitutive Cauliflower Mosaic Virus 35S promoter (pCaMV35S). Notably, pGhERF105 and pGhNc-HARBI1 promoters demonstrated more efficiency in driving reporter genes in flowers than other previously characterized cotton flower-specific promoters. Overall, the present study provides a new set of cotton promoters suitable for biotechnological application in cotton plants for pest resistance.

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.

Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil α-amylase.

BACKGROUND: Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis α-amylase (AGA). RESULTS: We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. CONCLUSIONS: We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.

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.

Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis).

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of ß-pore-forming toxins (ß-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

Transcription profile of soybean-root-knot nematode interaction reveals a key role of phythormones in the resistance reaction.

BACKGROUND: Root-knot nematodes (RKN- Meloidogyne genus) present extensive challenges to soybean crop. The soybean line (PI 595099) is known to be resistant against specific strains and races of nematode species, thus its differential gene expression analysis can lead to a comprehensive gene expression profiling in the incompatible soybean-RKN interaction. Even though many disease resistance genes have been studied, little has been reported about phytohormone crosstalk on modulation of ROS signaling during soybean-RKN interaction. RESULTS: Using 454 technology to explore the common aspects of resistance reaction during both parasitism and resistance phases it was verified that hormone, carbohydrate metabolism and stress related genes were consistently expressed at high levels in infected roots as compared to mock control. Most noteworthy genes include those encoding glycosyltransferases, peroxidases, auxin-responsive proteins and gibberellin-regulated genes. Our data analysis suggests the key role of glycosyltransferases, auxins and components of gibberellin signal transduction, biosynthesis and deactivation pathways in the resistance reaction and their participation in jasmonate signaling and redox homeostasis in mediating aspects of plant growth and responses to biotic stress. CONCLUSIONS: Based on this study we suggest a reasonable model regarding to the complex mechanisms of crosstalk between plant hormones, mainly gibberellins and auxins, which can be crucial to modulate the levels of ROS in the resistance reaction to nematode invasion. The model also includes recent findings concerning to the participation of DELLA-like proteins and ROS signaling controlling plant immune or stress responses. Furthermore, this study provides a dataset of potential candidate genes involved in both nematode parasitism and resistance, which can be tested further for their role in this biological process using functional genomics approaches.

Overexpression of the CaHB12 transcription factor in cotton (Gossypium hirsutum) improves drought tolerance.

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.

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.

RNAi-Mediated Suppression of Laccase2 Impairs Cuticle Tanning and Molting in the Cotton Boll Weevil (Anthonomus grandis).

The cotton boll weevil, Anthonomus grandis, is the most economically important pest of cotton in Brazil. Pest management programs focused on A. grandis are based mostly on the use of chemical insecticides, which may cause serious ecological impacts. Furthermore, A. grandis has developed resistance to some insecticides after their long-term use. Therefore, alternative control approaches that are more sustainable and have reduced environmental impacts are highly desirable to protect cotton crops from this destructive pest. RNA interference (RNAi) is a valuable reverse genetics tool for the investigation of gene function and has been explored for the development of strategies to control agricultural insect pests. This study aimed to evaluate the biological role of the Laccase2 (AgraLac2) gene in A. grandis and its potential as an RNAi target for the control of this insect pest. We found that AgraLac2 is expressed throughout the development of A. grandis with significantly higher expression in pupal and adult developmental stages. In addition, the immunolocalization of the AgraLac2 protein in third-instar larvae using specific antibodies revealed that AgraLac2 is distributed throughout the epithelial tissue, the cuticle and the tracheal system. We also verified that the knockdown of AgraLac2 in A. grandis resulted in an altered cuticle tanning process, molting defects and arrested development. Remarkably, insects injected with dsAgraLac2 exhibited defects in cuticle hardening and pigmentation. As a consequence, the development of dsAgraLac2-treated insects was compromised, and in cases of severe phenotypic defects, the insects subsequently died. On the contrary, insects subjected to control treatments did not show any visible phenotypic defects in cuticle formation and successfully molted to the pupal and adult stages. Taken together, our data indicate that AgraLac2 is involved in the cuticle tanning process in A. grandis and may be a promising target for the development of RNAi-based technologies.

Meloidogyne incognita: molecular cloning and characterization of a cDNA encoding a cathepsin D-like aspartic proteinase.

Herein we describe the cloning and characterization of a cDNA encoding an aspartic proteinase from the root-knot nematode Meloidogyne incognita. Using PCR techniques, a 1471-bp cDNA fragment encoding a cathepsin D-like (Mi-asp1) transcript was isolated from second-stage larvae mRNA. Its predicted amino acid sequence comprises a pro-region of 71 amino acid residues and a mature protease of 378 amino acid residues with a predicted molecular mass of 41.502kDa. Protein sequence comparisons of Mi-asp1 with GenBank (DQ360827) sequences showed 59-71% identity with nematode-specific cathepsin D-like aspartic proteinases. Southern blot analysis, RT-PCR amplification and EST mining suggest the existence of a developmentally expressed gene family encoding aspartic proteinases in M. incognita. Mi-asp1 may represent a potential target for molecular intervention for the purposes of plant-parasitic nematode control.

A recombinant form of chagasin from Trypanosoma cruzi: inhibitory activity on insect cysteine proteinases.

BACKGROUND: The activity of the major digestive cysteine proteinase detected in the intestinal tract of larvae of the bean weevil, Acanthoscelides obtectus (Say), was efficiently inhibited by the well-characterized cysteine proteinase synthetic inhibitor E-64 and also by a recombinant form of chagasin (r-chagasin), a tight-binding cysteine proteinase inhibitor protein from Trypanosoma cruzi. RESULTS: Incorporation of r-chagasin into an artificial diet system at 0.1 g kg(-1) retarded growth rate, decreased larval survival and led to complete mortality of A. obtectus at the end of the trial. The observed differences in growth rates occurred particularly in the first and second development stages. Artificial seeds containing high levels of r-chagasin (0.5-30 g kg(-1)) completely inhibited larval penetration. CONCLUSION: Together, the results reported in this paper support the hypothesis that the inhibitory activity of r-chagasin towards the major insect gut cysteine proteinase in vitro and in vivo is an accurate prediction of its insecticidal effects. The selectivity of this inhibitor against insect digestive proteinases supports the key role in parasite virulence by affecting the endogenous proteinase activity in its natural host.

Effect of a Bowman-Birk proteinase inhibitor from Phaseolus coccineus on Hypothenemus hampei gut proteinases in vitro.

The coffee berry borer, Hypothenemus hampei (Ferrari), is an important devastating coffee pest worldwide. Both trypsin and chymotrypsin enzyme activities from H. hampei larval midgut can be inactivated by proteinaceous enzyme-inhibitors. A serine proteinase inhibitor belonging to the Bowman-Birk class was purified from a wild accession of Phaseolus coccineus L. seeds. The inhibitor (PcBBI1) is a cysteine-rich protein that is heat-stable at alkaline pH. MALDI-TOF/MS analysis showed that PcBBI1 occurs in seeds as a monomer (8689 Da) or dimer (17,378 Da). Using in vitro inhibition assays, it was found that PcBBI1 has a high inhibitory activity against H. hampei trypsin-like enzymes, bovine pancreatic chymotrypsin, and trypsin. According to this, PcBBI1 could be a promising tool to make genetically modified coffee with resistance to coffee berry borer.

An alpha-amylase inhibitor gene from Phaseolus coccineus encodes a protein with potential for control of coffee berry borer (Hypothenemus hampei).

Plant alpha-amylase inhibitors are proteins found in several plants, and play a key role in natural defenses. In this study, a gene encoding an alpha-amylase inhibitor, named alphaAI-Pc1, was isolated from cotyledons of Phaseolus coccineus. This inhibitor has an enhanced primary structure to P. vulgaris alpha-amylase inhibitors (alpha-AI1 and alpha-AI2). The alphaAI-Pc1 gene, constructed with the PHA-L phytohemaglutinin promoter, was introduced into tobacco plants, with its expression in regenerated (T0) and progeny (T1) transformant plants monitored by PCR amplification, enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis, respectively. Seed protein extracts from selected transformants reacted positively with a polyclonal antibody raised against alphaAI-1, while no reaction was observed with untransformed tobacco plants. Immunological assays showed that the alphaAI-Pc1 gene product represented up to 0.05% of total soluble proteins in T0 plants seeds. Furthermore, recombinant alphaAI-Pc1 expressed in tobacco plants was able to inhibit 65% of digestive H. hampei alpha-amylases. The data herein suggest that the protein encoded by the alphaAI-Pc1 gene has potential to be introduced into coffee plants in order to increase their resistance to the coffee berry borer.

The recombinant pea defensin Drr230a is active against impacting soybean and cotton pathogenic fungi from the genera Fusarium, Colletotrichum and Phakopsora.

Plant defensins are antifungal peptides produced by the innate immune system plants developed to circumvent fungal infection. The defensin Drr230a, originally isolated from pea, has been previously shown to be active against various entomopathogenic and phytopathogenic fungi. In the present study, the activity of a yeast-expressed recombinant Drr230a protein (rDrr230a) was tested against impacting soybean and cotton fungi. First, the gene was subcloned into the yeast expression vector pPICZαA and expressed in Pichia pastoris. Resulting rDrr230a exhibited in vitro activity against fungal growth and spore germination of Fusarium tucumaniae, which causes soybean sudden death syndrome, and against Colletotrichum gossypii var. cephalosporioides, which causes cotton ramulosis. The rDrr230a IC50 corresponding to inhibition of fungal growth of F. tucumaniae and C. gossypii var. cephalosporioides was 7.67 and 0.84 µM, respectively, demonstrating moderate activity against F. tucumaniae and high potency against C. gossypii var. cephalosporioides. Additionally, rDrr230a at 25 ng/µl (3.83 µM) resulted in 100 % inhibition of spore germination of both fungi, demonstrating that rDrr230a affects fungal development since spore germination. Moreover, rDrr230a at 3 µg/µl (460.12 µM) inhibited 100 % of in vitro spore germination of the obligatory biotrophic fungus Phakopsora pachyrhizi, which causes Asian soybean rust. Interestingly, rDrr230a substantially decreased the severity of Asian rust, as demonstrated by in planta assay. To our knowledge, this is the first report of a plant defensin active against an obligatory biotrophic phytopathogenic fungus. Results revealed the potential of rDrr230a as a candidate to be used in plant genetic engineering to control relevant cotton and soybean fungal diseases.

Sugarcane giant borer transcriptome analysis and identification of genes related to digestion.

Sugarcane is a widely cultivated plant that serves primarily as a source of sugar and ethanol. Its annual yield can be significantly reduced by the action of several insect pests including the sugarcane giant borer (Telchin licus licus), a lepidopteran that presents a long life cycle and which efforts to control it using pesticides have been inefficient. Although its economical relevance, only a few DNA sequences are available for this species in the GenBank. Pyrosequencing technology was used to investigate the transcriptome of several developmental stages of the insect. To maximize transcript diversity, a pool of total RNA was extracted from whole body insects and used to construct a normalized cDNA database. Sequencing produced over 650,000 reads, which were de novo assembled to generate a reference library of 23,824 contigs. After quality score and annotation, 43% of the contigs had at least one BLAST hit against the NCBI non-redundant database, and 40% showed similarities with the lepidopteran Bombyx mori. In a further analysis, we conducted a comparison with Manduca sexta midgut sequences to identify transcripts of genes involved in digestion. Of these transcripts, many presented an expansion or depletion in gene number, compared to B. mori genome. From the sugarcane giant borer (SGB) transcriptome, a number of aminopeptidase N (APN) cDNAs were characterized based on homology to those reported as Cry toxin receptors. This is the first report that provides a large-scale EST database for the species. Transcriptome analysis will certainly be useful to identify novel developmental genes, to better understand the insect's biology and to guide the development of new strategies for insect-pest control.

Employing in vitro directed molecular evolution for the selection of α-amylase variant inhibitors with activity toward cotton boll weevil enzyme.

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 108 α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control.