Evidence of population expansion and insecticide resistance mechanism in invasive fall armyworm (Spodoptera frugiperda).

BACKGROUND: The invasive and calamitous polyphagous pest Spodoptera frugiperda or commonly known as fall armyworm (FAW) poses serious menace to the global agricultural production. Owing to the revamped invasion of FAW in 2018 in India, present study was undertaken for precise assessment of its genetic identity and pesticide resistance to aid in pest-management strategies. RESULTS: To evaluate the diversity in FAW population across Eastern India, mitochondrial COI sequences were used which revealed a low nucleotide diversity. Analysis of molecular variance indicated significant genetic variation between four global geographical FAW populations with lowest differentiation between India and Africa suggesting a present-day and shared origin of FAW. The study demonstrated existence of two different strains ('R' strain and 'C' strain) based on COI gene marker. However, discrepancies between COI marker and host plant association of FAW was observed. Characterization of Tpi gene revealed abundance of TpiCa1a followed by TpiCa2b and TpiR1a strains respectively. The FAW population showed higher susceptibility towards chlorantraniliprole and spinetoram than cypermethrin. Insecticide resistance genes depicted marked upregulation although with lot of variance. Chlorantraniliprole resistance ratio (RR) exhibited significant correlation with 1950 (Glutathione S-transferase, GST), 9131 (Cytochrome P450, CYP) and 9360 (CYP) genes, while spinetoram and cypermethrin RR was found to correlate with 1950 (GST) and 9360 (CYP) genes. CONCLUSION: This study manifests Indian subcontinent as the potential new hotspot for the growth and distribution of FAW population that can be effectively controlled using chlorantraniliprole and spinetoram. This study also adds novel significant information on FAW population across Eastern India for developing a comprehensive pest management approach for S. frugiperda.

The C-terminal stretch of glycine-rich proline-rich protein (SbGPRP1) from Sorghum bicolor serves as an antimicrobial peptide by targeting the bacterial outer membrane protein.

KEY MESSAGE: The C-terminal stretch in SbGPRP1 (Sorghum glycine-rich proline-rich protein) acts as an antimicrobial peptide in the host innate defense mechanism. Cationic antimicrobial proteins or peptides can either bind to the bacterial membrane or target a specific protein on the bacterial membrane thus leading to membrane perturbation. The 197 amino acid polypeptide of SbGPRP1 showed disordered structure at the N-terminal end and ordered conformation at the C-terminal end. In the present study, the expression of N-SbGPRP1, C-SbGPRP1, and ∆SbGPRP1 followed by antimicrobial assays showed potential antimicrobial property of the C-terminal peptide against gram-positive bacteria Bacillus subtilis and phytopathogen Rhodococcus fascians. The SbGPRP1 protein loses its antimicrobial property when the 23 amino acid sequence (GHGGHGVFGGGYGHGGYGHGYGG) from position 136 to 158 is deleted from the protein. Thus, it can be concluded that the 23 amino acid sequence is vital for the said antimicrobial property. NPN assay, SEM analysis, and electrolyte leakage assays showed potent antimicrobial activity for C-SbGPRP1. Overexpression of the C-SbGPRP1 mutant protein in tobacco followed by infection with Rhodococcus fascians inhibited bacterial growth as shown by SEM analysis. To determine if C-SbGPRP1 might target any protein on the bacterial membrane we isolated the bacterial membrane protein from both Bacillus subtilis and Rhodococcus fascians. Bacterial membrane protein that interacted with the column-bound C-SbGPRP1 was eluted and subjected to LC-MS/MS. LC-MS/MS data analysis showed peptide hit with membrane protein YszA from Bacillus subtilis and a membrane protein from Rhodococcus fascians. Isolated bacterial membrane protein from Bacillus subtilis or Rhodococcus fascians was able to reduce the antimicrobial activity of C-SbGPRP1. Furthermore, BiFC experiments showed interactions between C-SbGPRP1 and YszA protein from Bacillus subtilis leading to the conclusion that bacterial membrane protein was targeted in such membrane perturbation leading to antimicrobial activity.

Unraveling the Basis of Neonicotinoid Resistance in Whitefly Species Complex: Role of Endosymbiotic Bacteria and Insecticide Resistance Genes.

Bemisia tabaci (whitefly) is one of the most detrimental agricultural insect pests and vectors of many plant viruses distributed worldwide. Knowledge of the distribution patterns and insecticide resistance of this cryptic species is crucial for its management. In this study, genetic variation of mitochondrial cytochrome oxidase subunit 1 (MtCoI) gene of B. tabaci was analyzed followed by a study of the infection profile of various endosymbionts in 26 whitefly populations collected from West Bengal, India. Phylogenetic analysis revealed Asia I as the major cryptic species (65.38%), followed by Asia II 5, China 3, and Asia II 7, which were diversified into 20 different haplotypes. In addition to the primary endosymbiont (C. poriera), each of the four whitefly species showed a variable population of three secondary endosymbionts, majorly Arsenophonus with the highest infection rate (73.07%), followed by Wolbachia and Rickettsia. Further phylogenetic analyses revealed the presence of two subgroups of Arsenophonus, viz., A1 and A2, and one each in Wolbachia (W1) and Rickettsia (R3). Resistance to thiamethoxam, imidacloprid, and acetamiprid insecticides was analyzed for a clear picture of pesticide resistance status. The highest susceptibility was noted toward thiamethoxam (LC50 = 5.36 mg/L), followed by imidacloprid and acetamiprid. The whitefly population from Purulia and Hooghly districts bearing Asia II 7 and Asia II 5 cryptic species, respectively, shows maximum resistance. The differences in mean relative titer of four symbiotic bacteria among field populations varied considerably; however, a significant positive linear correlation was observed between the resistance level and relative titer of Arsenophonus and Wolbachia in the case of imidacloprid and thiamethoxam, while only Wolbachia was found in case of acetamiprid. Expression analysis demonstrated differential upregulation of insecticide resistance genes with Purulia and Hooghly populations showing maximally upregulated P450 genes. Moreover, thiamethoxam and imidacloprid resistance ratio (RR) showed a significant correlation with CYP6CM1, CYP6DZ7, and CYP4C64 genes, while acetamiprid RR correlated with CYP6CX1, CYP6DW2, CYP6DZ7, and CYP4C64 genes. Taken together, these findings suggested that P450 mono-oxygenase and symbiotic bacteria together affected whitefly resistance to neonicotinoids. Hence, a symbiont-oriented management programme could be a better alternative to control or delay resistance development in whitefly and can be used for pesticide clean-up in an agricultural field.

NBS1 protein from Physcomitrium patens confers protection against oxidative damage by limiting the accumulation of cellular reactive oxygen species.

Nijmegen breakage syndrome 1 (NBS1) protein is a core member of the MRE11-RAD50-NBS1 (MRN) complex that plays a crucial role in DNA damage sensing and repair in plants. Here we report that NBS1 from moss Physcomitrium patens reduces oxidative damage by lowering the cellular ROS in addition to its known role in oxidative DNA damage recovery. Real-time transcript analysis showed up-regulation of the PpNBS1 transcript under different stress conditions. Bacterial cells showed better cell survivability upon over-expressing PpNBS1 protein as compared to untransformed cells. Likewise, overexpression of PpNBS1 in tobacco plants provides improved protection against oxidative damage and exhibited a lesser amount of ROS upon exposure to oxidative stress. Moreover, PpNBS1 contributes to the antioxidant defense mechanism by positively regulating the expression of the antioxidant genes under stress conditions in transgenic tobacco plants. PpNBS1 expressing transgenic tobacco plants resulted in lesser membrane damage, lower lipid peroxidation level, and higher chlorophyll content under stress conditions. Taken together, we conclude in addition to its known role as DNA damage sensor, PpNBS1 also plays a definite role in oxidative stress mitigation by minimizing ROS accumulation in the cell.

A rice R2R3-MYB (OsC1) transcriptional regulator improves oxidative stress tolerance by modulating anthocyanin biosynthesis.

The R2R3 type MYB transcription factors participate in controlling flavonoid production in plants, including anthocyanin and proanthocyanin. Black rice with high anthocyanin content is an important candidate for understanding R2R3-MYB-based regulation of the anthocyanin biosynthesis pathway (ABP). This study was undertaken to draw the functional relationship of an R2R3-MYB protein with anthocyanin biosynthesis and oxidative stress tolerance in plants. The expression levels of the late ABP genes in the panicle stage of black rice were in good agreement with the accumulation of anthocyanin, especially cyanidin 3-glucoside. Among all MYB genes present in rice, an R2R3 type (C1) regulates anthocyanin biosynthesis and was studied further. The positive correlation between the expression of ABP genes and OsC1 along with the nuclear localization of OsC1 are in line with its possible involvement as a transcriptional regulator of ABP genes. Interestingly, OsC1 overexpressed in white rice plants triggered anthocyanin production through augmentation of the transcript level of late ABP genes. Moreover, OsC1-transformed plants exhibited a lower amount of reactive oxygen species upon exposure to oxidative stress. The increased anthocyanin content in white rice seedlings resulted in higher photosynthetic efficiency, less membrane damage and consequently lower oxidative stress. The OsC1 transcriptional regulator helps to ameliorate oxidative stresses in plants owing to its anthocyanin modulating ability.

Multiple copies of a novel amphipathic α-helix forming segment in Physcomitrella patens dehydrin play a key role in abiotic stress mitigation.

Plants use a diverse set of proteins to mitigate various abiotic stresses. The intrinsically disordered protein dehydrin is an important member of this repertoire of proteins, characterized by a canonical amphipathic K-segment. It can also contain other stress-mitigating noncanonical segments-a likely reflection of the extremely diverse nature of abiotic stress encountered by plants. Among plants, the poikilohydric mosses have no inbuilt mechanism to prevent desiccation and therefore are likely to contain unique noncanonical stress-responsive motifs in their dehydrins. Here we report the recurring occurrence of a novel amphipathic helix-forming segment (D-segment: EGφφD(R/K)AKDAφ, where φ represents a hydrophobic residue) in Physcomitrella patens dehydrin (PpDHNA), a poikilohydric moss. NMR and CD spectroscopic experiments demonstrated the helix-forming tendency of the D-segment, with the shuffled D-segment as control. PpDHNA activity was shown to be size as well as D-segment dependent from in vitro, in vivo, and in planta studies using PpDHNA and various deletion mutants. Bimolecular fluorescence complementation studies showed that D-segment-mediated PpDHNA self-association is a requirement for stress abatement. The D-segment was also found to occur in two rehydrin proteins from Syntrichia ruralis, another poikilohydric plant like P. patens. Multiple occurrences of the D-segment in poikilohydric plant dehydrins/rehydrins, along with the experimental demonstration of the role of D-segment in stress abatement, implies that the D-segment mediates unique resurrection strategies, which may be employed by plant dehydrins that are capable of mitigating extreme stress.

Glycine rich proline rich protein from Sorghum bicolor serves as an antimicrobial protein implicated in plant defense response.

KEY MESSAGE: Sorghum glycine rich proline rich protein (SbGPRP1) exhibit antimicrobial properties and play a crucial role during biotic stress condition. Several proteins in plants build up the innate immune response system in plants which get triggered during the occurrence of biotic stress. Here we report the functional characterization of a glycine-rich proline-rich protein (SbGPRP1) from Sorghum which was previously demonstrated to be involved in abiotic stresses. Expression studies carried out with SbGPRP1 showed induced expression upon application of phytohormones like salicylic acid which might be the key in fine-tuning the expression level. Upon challenging the Sorghum plants with a compatible pathogen the SbGprp1 transcript was found to be upregulated. SbGPRP1 encodes a 197 amino acid polypeptide which was bacterially-expressed and purified for in vitro assays. Gram-positive bacteria like Bacillus and phytopathogen Rhodococcus fascians showed inhibited growth in the presence of the protein. The NPN assay, electrolytic leakage and SEM analysis showed membrane damage in bacterial cells. Ectopic expression of SbGPRP1 in tobacco plants led to enhanced tolerance towards infection caused by R. fascians. Though the N-terminal part of the protein showed disorderness the C-terminal end was quite capable of forming several α-helices which was correlated with CD spectroscopic analysis. Here, we have tried to determine the structural model for the protein and predicted the association of antimicrobial activity with the C-terminal region of the protein.

Dehydrins Impart Protection against Oxidative Stress in Transgenic Tobacco Plants.

Environmental stresses generate reactive oxygen species (ROS) which might be detrimental to the plants when produced in an uncontrolled way. However, the plants ameliorate such stresses by synthesizing antioxidants and enzymes responsible for the dismutation of ROS. Additionally, the dehydrins were also able to protect the inactivation of the enzyme lactate dehydrogenase against hydroxyl radicals (OH⋅) generated during Fenton's reaction. SbDhn1 and SbDhn2 overexpressing transgenic tobacco plants were able to protect against oxidative damage. Transgenic tobacco lines showed better photosynthetic efficiency along with high chlorophyll content, soluble sugar and proline. However, the malonyl dialdehyde (MDA) content was significantly lower in transgenic lines. Experimental evidence demonstrates the protective effect of dehydrins on electron transport chain in isolated chloroplast upon methyl viologen (MV) treatment. The transgenic tobacco plants showed significantly lower superoxide radical generation () upon MV treatment. The accumulation of the H2O2 was also lower in the transgenic plants. Furthermore, in the transgenic plants the expression of ROS scavenging enzymes was higher compared to non-transformed (NT) or vector transformed (VT) plants. Taken together these data, during oxidative stress dehydrins function by scavenging the () directly and also by rendering protection to the enzymes responsible for the dismutation of () thereby significantly reducing the amount of hydrogen peroxides formed. Increase in proline content along with other antioxidants might also play a significant role in stress amelioration. Dehydrins thus function co-operatively with other protective mechanisms under oxidative stress conditions rendering protection in stress environment.

YSK2 Type Dehydrin (SbDhn1) from Sorghum bicolor Showed Improved Protection under High Temperature and Osmotic Stress Condition.

YSK2 type dehydrin from Sorghum bicolor (SbDhn1) showed a high level of transcript accumulation when subjected to high temperature and osmotic stress. The high transcript level occurring in such stress situation might lead to a protective effect; though the exact mechanism by which this is achieved remains poorly understood. Nevertheless, our results provide compelling evidence to prove that transgenic tobacco lines overexpressing SbDhn1 gene showed improve stress tolerance as assessed by reduced membrane damage and low MDA content. Furthermore, we demonstrate here SbDhn1 expressing lines were only able to recover after stress treatment. In this study, we have provided direct evidence for the protection rendered by SbDHN1 protein to a temperature-sensitive enzyme under both high temperature and osmotic stress. We extended this analysis to the whole plant proteome where the addition of SbDHN1 protein helped in retaining the solubility of the protein was demonstrated. Interestingly, in vitro experiments carried out with lactate dehydrogenase (LDH), showed aggregate formation upon subjecting it to high temperature. However, in presence of SbDHN1 protein very few aggregates were observed. Aggregation assay showed a high level of aggregates in wild-type or empty vector transformed plants as compared to SbDhn1 transgenic lines. Confocal microscopy images in leaf peel sections of wild-type plants showed high amounts of aggregates as compared with transgenic lines. This study provides evidence for the protection rendered by SbDHN1 protein under high temperature by inhibiting the aggregate formation and provide the rational for the mechanism how these proteins ameliorate the adverse stress conditions.

Different dehydrins perform separate functions in Physcomitrella patens.

MAIN CONCLUSION: Dehydrins, PpDHNA and PpDHNB from Physcomitrella patens provide drought and cold tolerance while PpDHNC shows antimicrobial property suggesting different dehydrins perform separate functions in P. patens. The moss Physcomitrella patens can withstand extremes of environmental condition including abiotic stress such as dehydration, salinity, low temperature and biotic stress such as pathogen attack. Osmotic stress is inflicted under both cold and drought stress conditions where dehydrins have been found to play a significant protective role. In this study, a comparative analysis was drawn for the three dehydrins PpDHNA, PpDHNB and PpDHNC from P. patens. Our data shows that PpDHNA and PpDHNB play a major role in cellular protection during osmotic stress. PpDHNB showed several fold upregulation of the gene when P. patens was subjected to cold and osmotic stress in combination. PpDHNA and PpDHNB provide protection to enzyme lactate dehydrogenase under osmotic as well as freezing conditions. PpDHNC possesses antibacterial activity and thus may have a role in biotic stress response. Overexpression of PpDHNA, PpDHNB and PpDHNC in transgenic tobacco showed a better performance for PpDHNB with respect to cold and osmotic stress. These results suggest that specific dehydrins contribute to tolerance of mosses under different stress conditions.