Geminiviruses infect a broad range of economically important crop species all over the world and are increasingly being shown to occur in mixed infections, resulting in synergistic interactions, characterized by severe symptoms and huge yield losses. Partly because of mixed infections, these viruses undergo recombination, presumably during replication, resulting in more virulent strains than their pedigree. One of the crops severely affected by these viruses is cassava (Manihot esculenta Crantz), a root crop that serves as a staple food to more than 500 million people worldwide. This new cassava virus, named East African cassava mosaic Cameroon virus (EACMCV) was isolated from cassava fields in Cameroon. We have shown that the EACMCV encoded AC4 protein is a symptom determinant and suppresses virus induced gene silencing (VIGS), an antivirus mechanism exhibited by eukaryotic plants. These studies also showed that AC4 targets the plasma and cytosolic membranes. Mutantional analyses showed that these properties depend on the AC4 encoded N-myristoylation motif, a membrane-binding motif, suggesting, that to function, AC4 must be within a membrane locale. These unique features and its small size (<10 kDa) make AC4 a potentially good probe to dissect and elucidate physiological and biochemical events occurring within the plant cell membrane following virus infection. The specific aims of this project are to: 1) Determine the role of AC4 protein in EACMCV pathogenesis, 2) Investigate the interaction between AC4 and the proteins that have been identified in a yeast two-hybrid screen, and 3) Functionally characterize the AC4-interacting proteins.
Since the plasma membrane plays an important role in trafficking in and out of the cell, AC4 likely plays an important role in virus entry and/or exit. This project will identify and characterize membrane proteins that interact with AC4 since such proteins might play a role in geminivirus infection, which would be crucial as new approaches are sought to control these viruses. An important component of this project is the training of undergraduate students, mostly from underrepresented minority groups, in molecular biology and biotechnology. This training will make these students competitive in the fast growing biotechnology job market upon graduation.
(NSF #0724083) As most viruses, geminiviruses have very few genes and therefore depend heavily on the host for replication and spread. In addition, viral genes encode multifunctional proteins. In this study, we investigated the AC4 protein encoded by one of eight genes of a cassava geminivirus isolated from Cameroon in West Africa. To determine whether AC4 is required for virus infection, mutations were introduced into conserved amino acid residues along the protein sequence and the infectivity of the mutant viruses assessed on Nicotiana benthamiana, an experimental host of cassava geminiviruses. Results showed that whereas this protein is not required for virus infection per se, mutations in specific sites cause led to additional mutations in the viral sequence (second site mutations), which either render the virus uninfectious or caused attenuated infections. Two of the mutations resulted in a reverse mutation to the wild-type (i.e. the virus reverted to the original sequence). This observation suggested that these viruses undergo frequent mutations, similar to RNA viruses. We therefore investigated genetic variation of the virus in naturally infected cassava and from experimentally infected N. benthamiana. Results showed that the populations of EACMCV in cassava and in N. benthamiana were genetically heterogeneous. Mutation frequencies in the order of 10-4, comparable to that reported for plant RNA viruses, were observed in both hosts. This is direct experimental evidence showing that cassava geminiviruses exhibit a high mutation frequency and that a single clone quickly transforms into a collection of mutant sequences upon introduction into the host. These results provide clear evidence of evolution in these viruses and explain the emergence of new and more virulent species as has been reported for geminiviruses infecting especially tomato, cotton, and cassava. Because we previously showed that AC4 binds to the plasma membrane and is a pathogenicity determinant, we investigated host proteins that interact with this protein using a yeast split-ubiquitin membrane protein two-hybrid screen of an Arabidopsis cDNA library. We have a large library (over 200) of putative AC4 interacting proteins. Most of these proteins have been sequenced and found to have different functions in the plant. Some examples of these proteins are: CARBONIC ANHYDRASE β, which involved in C3 plants’ host defense against infection; TUBULIN BETA CHAIN 2; DELTA TONOPLAST INTEGRAL PROTEIN, functions as a water channel and ammonium (NH3) transporter; A20/AN1-LIKE ZINC FINGER PROTEIN, which is a DNA binding, zinc ion binding protein with an unknown function. These proteins are being investigated to identify those involved in virus pathogenicity. AC4 was also shown to be a highly expressed protein in N. benthamiana, a plant routinely used for vaccine and other pharmaceutical production. We showed that this high expression is as a result of the AC4 encoded N-myristoylation encoded domain. We are therefore using this domain to improve vaccine production in plants. Over twenty students, most of them from minority groups traditionally underrepresented in the sciences, were trained during the execution of this project. These students acquired skills in molecular biology, virology and biotechnology and most of them either found jobs in the biotech sector or went on to attend graduate school.
