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Phase 4 (2009 Deadline)
Characterization and Utilization of Gossypium arboreum as a Source of Resistance Against Begomoviruses that are a Threat to the Cotton Crop in Pakistan and the United States
Peng W. Chee, University of Georgia
Shahid Mansoor, National Institute for Biotechnology and Genetic Engineering
Pakistani Funding (HEC): $106,000
US Funding (Department of State): $115,522
Project Dates: July 18, 2011 - July 18, 2014
Cotton leaf curl disease (CLCuD) complex is currently endemic throughout Pakistan and epidemic in Western India. Yield losses due to cotton leaf curl virus are as high as 100 percent in some cotton fields in Pakistan. Over the last ten years, the introduction of CLCuD resistant or tolerant cotton varieties in Pakistan produced by conventional breeding and selection methods has shown promise in helping manage the disease. However, in 2001 resistance was broken, with previously resistant varieties showing symptoms of CLCuD in the Vehari district of Pakistan, and in 2009-10 severe symptoms of cotton leaf curl disease appeared in several other districts. In this project, two cotton species resistant to cotton leaf curl disease complex will be crossed by the Cotton Research Institute in Faisalabad, a collaborating institution on the project, and the resulting plants will be tested for resistance to the disease. Scientists at NIBGE and the University of Georgia will use bioinformatics and molecular tools to identify polymorphism in genes that are involved in virus-host interactions such as those that interact with geminiviruses. This project should lay the foundation for addressing one of the most devastating diseases of cotton, which represents a shared concern of Pakistan (where it is already crippling cotton production in some areas) and the United States (where all germplasm appears to be vulnerable).
In 2013, the Pakistani PI Dr Shahid Mansoor, traveled to Co-PI Paterson Laboratory in Athens, Ga with funding from the Fulbright Program and established a RNA extraction procedure that works well for cotton. The RNA collected were sent to LC Sciences, which is based in Houston, TX and to the Boyce Thompson Institute at Cornell University for short RNA sequencing to determine the levels and diversity of short RNA (siRNA and miRNA) in G. arboreum
and G. herbaceum
. Preliminary analysis of miRNA isoforms miR482 (the most common miRNA that regulate R gene expressions) showed that this miRNA has many isoforms in G. arboreum
and G. herbaceum
, the two diploid species resistant to cotton leaf curl disease. In addition, the expression levels of miR482 were higher in different tissues of G. arboreum
as compared to G. hirsutum
. A comprehensive analysis of the two dataset is currently in progress.
Ms. Mychele Da Silva, a new PhD student, started working on this project. Since the cotton leaf curl virus is not found in the U.S., the team's approach to identify genes involved in plant disease-host interaction would require challenging the cotton plants with other disease causing pathogens such as root-knot nematodes, which is prevalent in all cotton production regions in the U.S. Ms. Da Silva has established a greenhouse inoculation procedure for the root-knot nematodes pathogen and will conduct an experiment to determine if resistance to nematode can be used as a model system to analyze the cotton genome for differences in R gene expression between the infected and non-infected (control) plants via sequencing.
The team have received seeds from different accessions of G. arboreum, G. herbaceum and G. gossypioides from the USDA cotton germplasm collection in College Station, TX. Plants from the three species above are currently growing in the greenhouse and will be cross-hybridized to develop genetic populations to dissect the genetics of resistance to cotton leaf curl virus.
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The Pakistani PI Dr. Mansoor, who is visiting the University of Georgia on a one-year Fulbright fellowship until September 2012, attended the Cotton Beltwide Conference in Orlando, Florida, in early January 2012. During the event, he and his US counterparts discussed their work in progress, as well as new findings related to resistance to cotton leaf curl disease complex. A consortium led by US co-PI Dr. Andrew Paterson recently completed the draft sequence of the G. raimondii
genome, available online at (http://www.phytozome.net/cotton.php
), which should help further the objectives of this joint project. The team reports that as of March 2012 they had obtained seeds of G. arboreum
and G. gossypioides
from the Cotton Germplasm Collection at College Station, Texas. The seeds have been planted in the greenhouses at the University of Georgia for cross hybridization in order to study the genetics of resistance to cotton leaf curl virus. Because the cotton leaf curl virus is not endemic to the United States, the researchers' approach to identifying genes involved in plant disease-host interaction will require challenging the cotton plants with other disease-causing pathogens. Dr. Mansoor has suggested an experimental approach using root-knot nematodes, a major pest that already affects cotton production in the United States. During the spring and summer of 2012, while Dr. Mansoor is still in Georgia on his fellowship, they will carry out the nematode challenge experiments and then analyze the genome for differences in gene expression between the infected and non-infected (control) plants via sequencing.
Thanks to an award from the Fulbright Visiting Scholar Program, the Pakistani PI Dr. Mansoor has been working in the lab of Dr. Chee’s co-PI Dr. Andrew Paterson at the University of Georgia since September 2011. Now that the genome of the model species G. raimondii has been completely sequenced, the PIs report that they now have more molecular tools available to understand the molecular basis of resistance in G. arboreum to cotton leaf curl disease and other pathogens. The team has found out that screening of BAC libraries of G. arboreum provides a convenient way to find disease resistance genes (R genes). In collaboration with Dr. Mansoor, the US team has been comparing the isoforms of micro RNAs between G. arboreum and G. hirsutum in order to determine the linkage between non-host resistance and the expression levels of R genes.
During the second half of 2012, the US lab continues to perform molecular sequencing of short RNAs (siRNA and miRNA) from the two cotton species showing resistance to cotton leaf curl disease. Preliminary analysis showed that miRNA has many isoforms and the expression levels were higher in different tissues of G. arboretum as compared to G. hirsutum. A new female PhD student (Mychele DaSilva) will begin her graduate assistantship on this project beginning in early August. In addition, a Pakistani PhD student, Shazia Rani, from Bahauddin Zakariya University, Multan, Pakistan will stay for 6 months at Dr. Chee’s laboratory. She is supported by the Higher Education Commission.
The Pakistani PI Dr. Mansoor returned to Pakistan in October 2012 after completing a one-year Fulbright fellowship at UGA and upon his return was reappointed as director of the Agriculture Biotechnology Division of NIBGE with HEC releasing funds to resume his work in Pakistan. While in Georgia, Dr. Mansoor recommended an experimental approach using root-knot nematodes, a major pest that already affects cotton production in the United States since cotton leaf curl virus is not endemic here. Next steps involve carrying out the nematode challenge experiments and then analyzing the genome for differences in gene expression between the infected and non-infected (control) plants via sequencing.
2013 Show summary || Hide summary || Download full report
2014 Show summary || Hide summary || Download full report