Differential expression of gut protein genes and population density of Arsenophonus contributes to sex-biased transmission of Bemisia tabaci vectored Cotton leaf curl virus

Whitefly, Bemisia tabaci (Gennadius) is an important pest of cotton causing direct damage as sap feeder and vector of Cotton leaf curl virus (CLCuV). Previous few studies suggest that female whiteflies are more efficient vector of begomovirusthan males, however the sex-biased transmission efficiency is still not clearly understood. Present studies with B. tabaci AsiaII-1 haplotype showed higher virus transmission efficiency of females compared to males. This variable begomovirus transmission efficiency has been related to previously identifiedkey factors associated with B. tabaci. The higher density of endosymbiont Arsenophonus and variable expression of some midgut proteins genes i.e. Cyclophilin, Knottin, Hsp40, Hsp70 may be possibly imparting higher vector competency to the females compared to males. The present studies suggest low abundance of Arsenophonus spp. as well as lower expressionof Cyclophilin genein males as compared to females. This is further supplemented by overexpression of Knottin, Hsp40, and Hsp70 genes in males compared to females and thus collectively all these factors might be playing a key role in low virus transmission efficiency of males. The relative density of Arsenophonus spp. and expression of midgut proteins genes in male and female whitefly first time enriches our understanding about sex-biased transmission efficiency of begomovirus.

Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.

Identifying Genetic Diversity of Cotton Leaf Curl Virus in Commercial Cotton (Gossypium hirsutum L.)

Plant viruses have become a major issue to the crop production around the world. These viruses have become an unavoidable limiting factor by causing rigorous crop deficit for those major crops which are economically important. Among these viruses, begomoviruses belonging to family Geminiviridae, have brought the major devastation to both monocots and dicots in tropical and sub-tropical regions. These begomoviruses are transmitted through white fly and cause diseases like mosaic, yellow mosaic, yellow leaf curling, and yellow vein etc. The objective of this study is to evaluate the association between the genetic diversity of begomoviruses and phenotypic data to identify the ideal cotton genotype for breeding. In this study, 30 leaf samples with CLCuD symptoms were analyzed using rolling circular amplification and PCR. Obtained results expressed the presence of begomovirus with its associated satellites (i.e. alpha and betasatellites) in six varieties, association of begomovirus and betasatellites in four varieties and combination of begomovirus with alphasatellite in six varieties. Severe disease symptoms were exhibited with these combinations in selected varieties but betasatellite-begomovirus complex demonstrated a deadly impact. Few varieties expressed the resistance against the begomoviruses, so these varieties can further be manipulated for breeding.

Gene Expression Evaluation of Multigenic Cotton (Gossypium hirsutum) against Cotton Leaf Curl Virus

One of the most crucial threats limiting the sustainable production of cotton is cotton leaf curl disease (CLCuD). There is dire need to produce a resistant variety that can combat CLCuD. For this purpose, virus resistant transgenic cotton plants (MNH-786) with C4 gene construct at T3 generation were selected and sown. Young fresh leaves of multigenic variety of MNH-786 were collected to confirm the transformed construct. Infected whiteflies were used for spreading on transgenic cotton MNH-786 variety with C4 construct to check percentage of infection. Whiteflies were collected from infected cotton plants showing CLCuD and reared in lab to increase the population of whiteflies. After 15 days of feeding, infected leaves of transgenic plants were collected and total DNA of infected leaves of transgenic cotton plant with virus load was extracted. At maturity, data of morphological characteristic was taken from the transgenic cotton plants of MNH-786 and control plants. Resistant transgenic cotton plants showed < 0.5% disease index and recorded more plant height in field condition. Total number of bolls per plant was 20% more in tolerant plants and 40% more in resistant plants as compared to susceptible plants. Molecular analysis of transgenic plants showed clear evidence that expression of construct 4 virus resistant gene against begomoviruses in resistant and tolerant group of transgenic plants was more as compared to susceptible group and control. © 2021 Friends Science Publishers

The Gossypium stocksii genome as a novel resource for cotton improvement

Cotton is an important textile crop whose gains in production over the last century have been challenged by various diseases. Because many modern cultivars are susceptible to several pests and pathogens, breeding efforts have included attempts to introgress wild, naturally resistant germplasm into elite lines. Gossypium stocksii is a wild cotton species native to Africa, which is part of a clade of vastly understudied species. Most of what is known about this species comes from pest resistance surveys and/or breeding efforts, which suggests that G. stocksii could be a valuable reservoir of natural pest resistance. Here we present a high-quality de novo genome sequence for G. stocksii. We compare the G. stocksii genome with resequencing data from a closely related, understudied species (G. somalense) to generate insight into the relatedness of these cotton species. Finally, we discuss the utility of the G. stocksii genome for understanding pest resistance in cotton, particularly resistance to cotton leaf curl virus.

Cotton leaf curl virus (leaf curl disease of cotton).

Leaf curl disease of cotton caused by the CLCuD-complex of begomoviruses is endemic to Pakistan and India and perhaps other nearby locales in south Asia. It has been introduced from there to China and the Philippines on ornamental plants, from where it has spread to infect cotton and okra in China. Losses are difficult to assess, but early estimates (pre-1990) range up to 20% when infection occurs early in the growing season and/or with highly susceptible cultivars. Viruliferous whiteflies on infested/infected plants harbouring CLCuD-begomoviruses imported to other cotton-growing countries, in particular, are of concern in preventing introduction under optimal circumstances. No seed transmission is known to occur.

The Gossypium stocksii genome as a novel resource for cotton improvement

Cotton is an important textile crop whose gains in production over the last century have been challenged by various diseases. Because many modern cultivars are susceptible to several pests and pathogens, breeding efforts have included attempts to introgress wild, naturally resistant germplasm into elite lines. Gossypium stocksii is a wild cotton species native to Africa, which is part of a clade of vastly understudied species. Most of what is known about this species comes from pest resistance surveys and/or breeding efforts, which suggests that G. stocksii could be a valuable reservoir of natural pest resistance. Here we present a high-quality de novo genome sequence for G. stocksii. We compare the G. stocksii genome with resequencing data from a closely related, understudied species (G. somalense) to generate insight into the relatedness of these cotton species. Finally, we discuss the utility of the G. stocksii genome for understanding pest resistance in cotton, particularly resistance to cotton leaf curl virus.

Cotton germplasm improvement and progress in Pakistan

Cotton (Gossypium spp.) contributes significantly to the economy of cotton-producing countries. Pakistan is the fourth-largest producer of cotton after China, the USA and India. The average yield of cotton is about 570.99 kg.hm− 2 in Pakistan. Climate change and different biotic stresses are causing reduction in cotton production. Transgenic approaches have unique advantage to tackle all these problems. However, how to confer permanent resistance in cotton against insects through genetic modification, is still a big challenge to address. Development of transgenic cotton has been proven to be effective. But its effectiveness depends upon several factors, including heterogeneity, seed purity, diffusion of varieties, backcrossing and ethical concerns. Cotton biotechnology was initiated in Pakistan in 1992–1993 with a focus on acquiring cotton leaf curl virus (CLCuV)-resistant insect-resistant, and improving fiber quality. This review summarizes the use of molecular markers, QTLs, GWAS, and gene cloning for cotton germplasm improvement, particularly in Pakistan.