Evaluation of BG, NPR1, and PAL in cotton plants through Virus Induced gene silencing reveals their role in whitefly stress.

Whitefly is one of the most hazardous insect pests that infests a wide range of host plants and causes huge damage to crop worldwide. In order to engineer plants resilient to whitefly stress, it is important to identify and validate the responsive genes by exploring the molecular dynamics of plants under stress conditions. In this study three genes BG, NPR1, and PAL genes have been studied in cotton for elucidating their role in whitefly stress response. Initially, insilico approach was utilized to investigate the domains and phylogeny of BG, NPR1 and PAL genes and found out that these genes showed remarkable resemblance in four cotton species Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii. In BG proteins the main functional domain was X8 belonging to glycohydro superfamily, in NPR1 two main functional domains were BTB_POZ at N terminal and NPR1_like_C at C terminal. In PAL functional domain PLN was found which belongs to Lyase class I superfamily. The promoter analysis of these genes displayed enrichment of hormone, stress and stimuli responsive cis elements. Through Virus Induced Gene Silencing (VIGS), these genes were targeted and kept under whitefly infestation. Overall, the whitefly egg and nymph production were observed 60-70% less on gene down regulated plants as compared to control plants. The qPCR-based expression analysis of certain stress-responsive genes showed that in BG down regulated plants the elevated expression of these whitefly responsive genes was detected, in NPR1 down regulated plants JAZ1 and HSP were found up regulated, ERF1 and WRKY40 didn't show significant differential expression, while MAPK6 was slightly down regulated. In PAL down regulated plants ERF1 and JAZ1 showed elevated expression while others didn't show significant alternation. Differential expression in gene down-regulated plants showed that whitefly responsive genes act in a complex inter signaling pathway and their expression impact each other. This study provides valuable insight into the structural and functional analysis of important whitefly responsive genes BG, NPR1, and PAL. The results will pave a path to future development of whitefly resilient crops.

Development of modified Cry1Ac for the control of resistant insect pest of cotton, Pectinophora gossypiella.

Cotton has been one of the most important cash crops in Pakistan, but its production is adversely affected by biotic and abiotic stresses. Insect pests such as pink bollworm present a colossal vulnerability to such a financially important commodity. Bt toxins have been widely used to safeguard agricultural plants against notorious insect pests such as cotton bollworm and pink bollworm, and they have proven to be effective in reducing chewing insect pests. However, its efficacy has been challenged due to the development of resistance in insect pests against Bt toxins such as Cry1Ac and this poses a significant risk to the long-term adoption of these Bt crops. Resistance in insect pests against Bt toxin Cry1Ac is developed due to the mutations in the midgut receptors such as cadherin. In this study first 56 amino acids which also includes helix alpha-1 portion from N-terminus of the Cry1Ac were removed and the gene was commercially synthesized following codon optimization. Modified Cry1Ac was used to develop transgenic plants of Nicotiana tabacum and insect bioassays were conducted to check the efficacy of Cry1Ac through leaf bioassays. Cry1Ac, a modified Bt toxin, was produced pET-28a (+), and diet bioassays were performed using purified protein at various doses against Pectinophora gossypiella. Based on the insect mortality and LC50, the Cry1AcM3 form of the modified toxins was shown to be more potent than the other modified versions (Cry1AcM1, Cry1AcM2), with more than 80 % mortality against resistant pink bollworm at 1.25 g/mL and an LC50 of 0.48. The results suggest that modified toxin cry1Ac may be useful in controlling population of pink bollworm resistant against cry1Ac.

Transcriptome diversity assessment of Gossypium arboreum (FDH228) leaves under control, drought and whitefly infestation using PacBio long reads.

Alternative splicing (AS) and alternative polyadenylation (APA) are common mechanisms in eukaryotes to increase the complexity of transcriptomes and subsequently proteomes. Analysis of long reads transcriptomics data can result in the discovery of novel transcripts, splice sites, AS or APA events. Gossypium arboreum is an important cultivated cotton species and a putative contributor of the A sub-genome to the modern tetraploid cotton; and inherently tolerant to several biotic and abiotic stresses. Specifically, its variety 'FDH228' is considered to be an important resistance source. In this study, we sequenced the G. arboreum (var. FDH228) transcriptome using PacBio IsoSeq and illumina short read sequencing under three different conditions i.e. untreated/healthy, treated with biotic stress through whitefly infestation, and treated with abiotic stress via water deprivation, for the discovery and surveying of canonical and non-canonical AS, APA and transcript fusion events. We were able to obtain 15,419 unique transcripts from all samples representing 11,343 genes, out of which 10,832 were annotated and 520 were novel with respect to the published reference genome. These transcripts were grouped into different structural categories including 60 Antisense, 11,959 having a full-splice match, 999 with incomplete-splice match, 30 fusion transcripts, 177 genic, 479 intergenic, 771 novels in the catalog, and 944 Novel but not found in the catalog. Subsequently, randomly selected candidate transcripts were experimentally validated using qRT-PCR. Our comprehensive identification of canonical and non-canonical splicing events, and novel and fusion transcripts aids in the understanding of the resistance mechanisms for this specific germplasm.

Cotton Mi-1.2-like Gene: A potential source of whitefly resistance.

Whitefly (Bemisia tabaci) inflicts tremendous yield losses to cotton crops in many parts of the world by sap-sucking and transmitting viral diseases. The tomato-associated Mi-1.2 gene has been successfully deployed in tomato cultivars to attain whitefly resistance. In the current study, putative Mi-1.2-like orthologs were identified in five whitefly hosts and functionally validated through virus-inducing gene silencing (VIGS) in cotton plants. The expression profiling and qPCR results depicted differential regulation of the Mi-1.2-like gene in various tissue types and under different biotic and abiotic stresses, especially in whitefly susceptible and resistant cotton plants. The upregulation of the Mi-1.2-like gene (Gadrp RPP-13 Like gene) was observed at 24 h and 48 h post-whitefly exposure (PWFE) in whitefly resistant (FDH-228) and tolerant (Mac7) cotton plants as compared to susceptible plants of Coker-312. However, delayed expression was recorded at 72 h of PWFE in Coker-312 plants. In TRV based gene silencing experiment, silencing of the Mi-1.2-like gene, significantly enhanced the whitefly infestation on both whitefly-resistant and susceptible cotton genotypes. Based on these results, we conducted the evolutionary analysis of Mi-1.2-like orthologs among cotton, cassava, tomato, papaya, and cucumber hosts. This indicated that cotton associated Mi-1.2 like gene has a close relation with cassava and tomato. These results suggested that Mi-1.2-like R genes could be the potential candidate for deriving whitefly resistance response in cotton plants.

Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae

BACKGROUND: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plantderived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. RESULTS: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. CONCLUSIONS: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloemspecific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.