Thursday, December 02, 2021
Molecular Virology and Gene Silencing

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Group Leader:

Dr. Shahid Mansoor, SI

Team Members:

 

Geminiviruses and their diversity in Pakistan

This group is pioneer in Pakistan who worked on molecular diversity of geminiviruses in Pakistan. The first epidemic of cotton leaf curl disease (CLCuD) in early 1990’s in the Indian subcontinent was associated with several distinct begomoviruses along with a disease-specific betasatellite. Resistant cotton varieties were introduced in late 1990’s but soon resistance was broken and was associated with a single recombinant begomovirus named Burewala strain of Cotton leaf curl Kokhran virus that lacks a full complement of a gene encoding a transcription activator protein (TrAP). In order to understand the ongoing changes in CLCuD complex in Pakistan, CLCuD affected plants from cotton fields at Vehari were collected. Illumina sequencing was used to assess the diversity of CLCuD complex. At least three distinct begomoviruses characterized from the first epidemic; Cotton leaf curl Multan virus, Cotton leaf curl Kokhran virus and Cotton leaf curl Alabad virus, several distinct species of alphasatellites and cotton leaf curl Multan betasatellite were found associated with CLCuD. These viruses were also cloned and sequenced through Sanger sequencing to confirm the identity of the begomoviruses and that all clones possessed a full complement of the TrAP gene. A new strain of betasatellite was identified here and named CLCuMuBVeh.

 

Diversity of whitefly biotypes in Pakistan

Bemisiatabaci (Gennadius; Hempitera: Aleyrodidae) is considered to be a cryptic (sibling) species complex, themembers of which exhibit morphological invariability while being genetically and behaviorally distinct. Membersof the complex are agricultural pests that cause direct damage by feeding on plants, and indirectly by transmittingviruses that cause diseases leading to reduced crop yield and quality. In Pakistan, cotton leaf curl disease, caused bymultiple begomovirus species, is the most economically important viral disease of cotton. In the study outlined here,the diversity and geographic distribution of B. tabaci cryptic species was investigated by analyzing a taxonomicallyinformative fragment of the mitochondrial cytochrome c oxidase 1 gene (mtCOI-3'). The mtCOI-3' sequence wasdetermined for 285 adult whiteflies and found to represent six cryptic species, the most numerous being Asia II-1and Middle East Asia Minor 1 (MEAM-1), the later also referred to as the B-biotype, which was previously thought tobe confined to Sindh province but herein, was also found to be present in the Punjab province. The endemic Asia Iwas restricted to Sindh province, while an individual in the Asia II-8 was identified in Pakistan for the first time. Alsofor the first time, samples were collected from northwestern Pakistan and Asia II-1 was identified. Results indicatethat in Pakistan the overall diversity of B. tabaci cryptic species is high and, based on comparisons with findingsfrom previous studies, the distribution is dynamic.

  

Virus induced gene silencing as a tool for functional genomics

This group has established theconditions for virus-induced gene silencing (VIGS) in threecultivated cotton species (Gossypium hirsutum, G. arboreum,andG. herbaceum) using a Tobacco rattle virus(TRV) vector. The system was used to silence the homologof the Arabidopsis thaliana chloroplastosalterados 1(AtCLA1) gene, involved in chloroplast development, inG. herbaceum, G. arboreum, and six commercial G. hirsutum cultivars. All plants inoculated with the TRV vectorto silence CLA1 developed a typical albino phenotypeindicative of silencing this gene. Although silencing in G.herbaceum and G. arboreum was complete, silencingefficiency differed for each G. hirsutum cultivar. Reversetranscriptase polymerase chain reaction (PCR) and real-timequantitative PCR showed a reduction in mRNA levelsof the CLA1 homolog in all three species, with the highestefficiency (lowest CLA1 mRNA levels) in G. arboreumfollowed by G. herbaceum and G. hirsutum. The resultsindicate that TRV is a useful vector for VIGS in Gossypiumspecies. This optimized method is now routinely used for the functional genomics of genes in cotton.

 

Precise CRISPR-Cas9 mediated genome editing in Super Basmati rice for resistance against bacterial blight by targeting the major susceptibility gene.  

Basmati rice is famous around the world for its flavor, aroma, and long grain. Its demand is increasing worldwide, especially in Asia. However, its production is threatened by various problems faced in the fields, resulting in major crop losses. One of the major problems is bacterial blight caused by Xanthomonasoryzaepv. oryzae (Xoo). Xoo hijacks the host machinery by activating the susceptibility genes (OsSWEET family genes), using its endogenous transcription activator like effectors (TALEs). TALEs have effector binding elements (EBEs) in the promoter region of the OsSWEET genes. Out of six well-known TALEs found to have EBEs in Clade III SWEET genes, four are present in OsSWEET14 gene’s promoter region. Thus, targeting the promoter of OsSWEET14 is very important for creating broad-spectrum resistance. To engineer resistance against bacterial blight, we established CRISPR-Cas9 mediated genome editing in Super Basmati rice by targeting 4 EBEs present in the promoter of OsSWEET14. We were able to obtain four different Super Basmati lines (SB-E1, SB-E2, SB-E3, and SB-E4) having edited EBEs of three TALEs (AvrXa7, PthXo3,  and TalF ). The edited lines were then evaluated in triplicate for resistance against bacterial blight by choosing one of the locally isolated virulent Xoo strains with AvrXa7  and infecting Super Basmati. The lines with deletions in EBE of AvrXa7 showed resistance against the Xoo  strain. Thus, it was confirmed that edited EBEs provide resistance against their respective TALEs present in Xoo strains. In this study up to 9% editing efficiency was obtained. Our findings showed that CRISPRCas9 can be harnessed to generate resistance against bacterial blight in indigenous varieties, against locally prevalent Xoo strains.

 

Genome editing of Potato

Potato (Solanum tubersom) belongs to Solanaceaefamily. It is fourth most important food crop after rice, wheat and maize,in term of food consumption. Its production rate is affected by abiotic like and biotic factors like bacteria, fungi, and viruses. Among viruses, PVY is most crop damaging strains with severe symptoms. Plant viruses encode a numbers of integral proteins while coding capacity might be limited by various host factors. Viruses highjack translation factors and other machinery of host for replication of viral RNAs. Usurped translation factors play critical role in translation of viral genome and helping viral movement in various parts of plants such as translation initiation factors like eIF4E and isoform eIF(iso)4E. Many plants have been attained resistance if these factor are either present in recessive form or mutated form. Studies at molecular level of virus-host interaction can decipher various plant genes that are crucial to viral infection and antiviral defenseAn RNA-programmed genome editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9,is most efficient SDM technique.CRISPR/Cas9 used to down regulate and manipulate the translation factors eIF4E, eIF(iso)4E and Vin of the host plant, the ability of PVY’s replication and interaction with host’s translation factors would be diminished and potato can be stored for longer period. Currently transformation is in process.

 

New plant breeding technologies for food security 

A world without hunger is possible but only if food production is sustainably increased and distributed and extreme poverty is eliminated. Globally, most of the poor and undernourished people live in rural areas of developing countries, where they depend on agriculture as a source of food, income, and employment. International data show a clear association between low agricultural productivity and high rates of undernourishment. Global studies have also shown that rapid reduction of extreme poverty is only possible when the incomes of smallholder farmers are increased. Therefore, sustained improvement in agricultural productivity is central to socioeconomic development. Here, we argue that with careful deployment and scientifically informed regulation, new plant breeding technologies (NPBTs) such as genome editing will be able to contribute substantially to global food security.

 

Virus-host interaction study

Begomoviruses interfere with host plant machinery to evade host defense mechanism by interacting with plant proteins. In the old world, this group of viruses are usually associated with betasatellite that induces severe disease symptoms by encoding a protein, βC1, which is a pathogenicity determinant. Here, we show that βC1 encoded by Cotton leaf curl Multan betasatellite (CLCuMB) requires Gossypium hirsutum calmodulin-like protein 11 (Gh- CML11) to infect cotton. First, we used the in silico approach to predict the interaction of CLCuMB-βC1 with Gh-CML11. A number of sequence- and structure-based in-silico interaction prediction techniques suggested a strong putative binding of CLCuMB-βC1 with Gh- CML11 in a Ca+2-dependent manner. In-silico interaction prediction was then confirmed by three different experimental approaches: The Gh-CML11 interaction was confirmed using CLCuMB-βC1 in a yeast two hybrid system and pull down assay. These results were further validated using bimolecular fluorescence complementation system showing the interaction in cytoplasmic veins of Nicotiana benthamiana. Bioinformatics and molecular studies suggested that CLCuMB-βC1 induces the overexpression of Gh-CML11 protein and ultimately provides calcium as a nutrient source for virus movement and transmission. This is the first comprehensive study on the interaction between CLCuMB-βC1 and Gh-CML11 proteins which provided insights into our understating of the role of βC1 in cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) caused by viruses of genus Begomovirus  is a major constraint to cotton (Gossypium hirsutum ) production in many cotton-growing regions of the world. Symptoms of the disease are caused by Cotton leaf curl Multan betasatellite (CLCuMB) that encodes a pathogenicity determinant protein, b C1. Here, we report the identification of interacting regions in b C1 protein by using computational approaches including sequence recognition, and binding site and interface prediction methods. We show the domain-level interactions based on the structural analysis of G. hirsutum SnRK1 protein and its domains with CLCuMB-b C1. To verify and validate the in silico predictions, three different experimental approaches, yeast two hybrid, bimolecular fluorescence complementation and pull down assay were used. Our results showedthat ubiquitin-associated domain (UBA) and autoinhibitory sequence (AIS) domains of G. hirsutum -encoded SnRK1 are involved in CLCuMB-b C1 interaction. This is the first comprehensive investigation that combined in silico  interaction prediction followed by experimental validation of interaction between CLCuMB-b C1 and a host protein. We demonstrated that data from computational biology could provide binding site information between CLCuD-associated viruses/satellites and new hosts that lack known binding site information for protein–protein interaction studies. Implications of these findings are discussed.

 

Computational Biology and livestock 

This group has state of the art facility for computational Biology and currently working on genomics and transcriptomics of plants and livestock. This group have completed whole genome sequencing of major cattle breeds in Pakistan.  Multiple projects are going on dealing with identification of markers for genomic selection of cattle breeds in Pakistan.

   
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