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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 (Extended through February 28, 2015)
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).
As the cotton leaf curl virus (CLCuV) is not endemic to the U.S., the research team is experimenting with an alternative method to study the expression of non-host resistance genes by infecting cotton plants with root-knot nematodes, which is a major pest of cotton production in the U.S and Pakistan. By using RNASeq gene expression analysis, the team aims to identify resistant genes that are responsive to the development of root-knot nematodes in cotton roots. The plan is to establish a healthy population of nematodes and continue with the experiment in January of 2015.
An alternative means to study non-host resistance genes is to compare CLCuV to other similar characteristic cotton viral diseases. Drs. Chee and Mansoor are now working collaboratively with Dr. Galbieri, a plant pathologist with the Instituto Mato Grossense de Algodao in Brazil, to determine if G. arboreum has the same non-compatible reaction or resistance to the cotton blue disease caused by the cotton leafroll dwarf virus. A preliminary test suggests that all genotypes were either resistant or moderately resistant to the cotton blue disease pathogen. Therefore, a comparison of the gene expression between G. arboreum plants infected with the cotton blue disease and the CLCuV may offer a means to identify non-host resistance genes. In this light, project staff will infect cotton plants with blue disease and the CLCuV then collect RNA samples from infected and control plants. The RNA samples will be sent to the Chee lab for sequencing, and data will be analyzed to identify a common set of resistant genes that are up or down regulated in the infected plants.
In terms of professional training, Ms. Mychele Da Silva, a graduate student funded by this project, has essentially completed most of her course work and is now working full time on her dissertation research involving this project on the UGA-Tifton Campus. Drs. Mansoor, Chee and Paterson attended the Biannual International Cotton Genome Initiative Conference, which was held in September in Wuhan, China. At the conference Dr. Paterson gave a plenary talk while Dr. Mansoor gave a presentation in the Cotton Leaf Curl Virus Workshop, and Dr. Chee presented a poster.
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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.