MOLECULAR CHARACTERIZATION OF A POLEROVIRUS INFECTING COTTON IN ALABAMA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1026345
• Project Start Date: Apr 28, 2021
• Project End Date: Mar 14, 2026
• Program Code: [(N/A)]- (N/A)

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
Plant Pathology

Non Technical Summary
Since its first report in 2017, Cotton leafroll dwarf virus (CLRDV) has spread across the cotton belt. Based on whole genome sequence analysis, the strain of CLRDV found in Alabama (CLRDV-AL) is genetically different from previously known strains. CLRDV-AL infects cotton genotypes resistant to formerly known 'typical' and 'atypical' strains and produces Cotton leafroll dwarf disease (CLRDD). Despite its potential threat, little is known about this virus as it is a newly introduced pathogen in the area. Identifying the possible variants of viral pathogen present in the mixed infection is crucial as multiple strains of the same virus interact and form particular dynamics, leading to exclusion or synergism that needs to be considered while developing a suitable management strategy against the disease. Also, it is crucial to investigate what makes the Alabama strain different from other CLRDV strains in successful infection in the host plant. Such distinction in pathogenicity can arise from a specific factor encoded in the virus and the host factor responding to a viral factor. This HATCH project aims to understand the complex virus population present in cotton-growing fields and dissect molecular and cellular characteristics of the viral pathogen at RNA and protein levels.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1710	1101	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1710 - Upland cotton;

Field Of Science
1101 - Virology;

Keywords

plant virus

polerovirus

cotton

pathogenicity

Goals / Objectives
The goal of this HATCH project is to harness tools in molecular and cellular biology to investigate the mechanism of poleroviral infection and contribute to the effective and sustainable management of viral disease. The specific objectives of this HATCH project are to 1) examine complex virus populations present in the crop growing in the fields, 2) identify pathogenicity factors encoded in the virus as well as associated host plant factors, 3) characterize the polerovirus prevalent in Alabama cotton fields at the RNA and protein levels. The project leader will seek funding for proposed research from the state cotton commodity board and major external sources such as USDA-NIFA and NSF for the components expecting field-applicable products and advancing the basic understanding of molecular virology, respectively.

Project Methods
Objective#1.Examine complex virus populations present in the plants growing on the fields.Identify the variants of CLRDV present in cotton-producing fields in Alabama. Samples for the analysis will be collected i) from the plants showing the symptoms of virus infection and transferred to the laboratory by extension specialists (Auburn University) for total RNA extraction and ii) by obtaining previously extracted and preserved total RNAs available at Plant Diagnostic Lab (Alabama Cooperative Extension System at Auburn). CLRDV will be amplified from total RNA using i) broad-spectrum primers designed to detect a wide range of different CLRDV strains and ii) specific primers designed to detect certain CLRDV strains. This will elucidate the presence of mixed infection by multiple strains of CLRDV in Alabama fields. The mixed infections of multiple CLRDV strains will be analyzed using a group of selected samples - identified to have complex populations - for the population composition and population ratio per strain using a quantitative amplification (q-PCR) method. This will clarify any exclusion or cross-protection among different strains shown by the population ratio of each strain present in the mixed infection. The contribution of CLRDV-AL in the mixed infection will be assessed by observing local and systemic symptom development on the plants in a laboratory setting and comparing how thepopulation ratio in mixed infection alters them to investigate the dynamics of the complex CLRDV population with emphasis on CLRDV-AL.Objective#2.identify pathogenicity factors encoded in the viral pathogen as well as the host factors in plants.Each CLRDV-encoded protein will be expressed individually to investigate its molecular and cellular characteristics. A set of clones of each gene encoded by the virus genome will be amplified with extra peptides tag and subsequently cloned into a binary vector for agrobacterium-mediated ectopic expression in the plants under the controlled environment within the laboratory. Upon agroinfiltration into the plants, the expression of each protein will be verified using an antibody against peptides-tag linked to the viral genes. The leaves infiltrated with an individual protein- expressing clone will be monitored for the development of virus-plant interaction, such as hypersensitive reaction in the infected area. The tissue of infiltrated area will be collected and processed for total RNA and total protein extraction to examine the expression level of specific host genes - i.e., pathogenicity-related genes - that have shown a correlated change in RNA level will be analyzed using quantitative amplification (q-PCR) method. This will elucidate potential host factors modulating a plant response upon poleroviral infection. Another set of clones expressing individual virus-encoded protein fused to fluorescence proteins will be generated for the observation using fluorescence microscopy. Examining the intracellular localization of viral proteins relative to the cellular organelles will provide clues about the potential mechanism of identified viral pathogenicity factors in the plants for successful infection.Objective#3.Characterize a polerovirus prevalent in Alabama cotton fields at the RNA and protein levels.The identified pathogenicity factors from CLRDV-AL will be compared with their equivalents from other CLRDV strains for the characteristics observed in Objective#2. The cognate proteins identified in Objectives#2 but encoded in different strains of CLRDV will be cloned as described above. Upon the expression of these proteins, the same parameters observed in Objectives#2 will be examined as well as the set of host genes analyzed for the plant response will be measured.The intracellular localization of viral proteins and their interaction between the pair of hetero- strain combinations - a protein from one strain and another from a different strain - will be investigated.

Progress 04/28/21 to 09/30/21

Outputs
Target Audience:The target audiences reached during this period were two groups, undergraduate students and Alabama cotton board. First, undergraduate students were invited to join the research by working on the experiments proposed in the project. Furthermore, PI created a course for these students (see Accomplishments section). Second, some proposedworks in the project was shared withAlabama Cotton Commission at the annual meeting. Changes/Problems:The majorproblem faced is the lack of full-timeresearcher. Two graduate students who were supposed to start in Spring and Fall, respectively, could not join the program due to the VISA issue related to COVID pandemic. Thus, the progress of proposed study has been slow. There will be one more undergraduate student joining the program and will work part time. Also, a new graduate student recuritment will be attempted for the following semester. What opportunities for training and professional development has the project provided?Two undergraduate students participated in the cloning of CLRDV genes for the plant expression through Undergraduate Research program offeredduring the summer (and continued into Fall semester). Students learned scientific research techniques by reading research papers and implementing hypothesis design, conducting experiments to validate it and interpretatingthe results from the experiments. Students also practiced on scientific writing and presentation. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The set of hybrid clones (CLRDV and fluorescence protein) will be completed and all will be introduced into the model plants for in planta examination to characterize eachviral proteins in CLRDV infection. First, the survey of plant response upon the expression of single CLRDV protein will be done using visual observation and gene expression analysis.Then, the examination of the intracellular localization CLRDV proteins will be followed using fluoroscence microscopy and in-situ hybridization.

Impacts
What was accomplished under these goals? A set of genes encoded within theCLRDV genome (P0, P1, P3, P4 and P3a) wasamplified and subsequentlycloned into binary vector with 35S promoter for plant expression. Some genes are in the process of cloning to add fluorescence protein gene (GFP and RFP) for further examination of CLRDV proteins in the plant cellsusing microscopy.

Publications