| One of the most commonly reported viral diseases of cowpea ( Vigna unguiculata, also referred to as blackeye pea) is cowpea mosaic disease, caused by Cowpea mosaic virus (CPMV). Various lines of cowpea differ in their degree of susceptibility to this virus, from supporting systemic infection to showing extreme resistance. One cowpea line, "Arlington," which shows extreme resistance to CPMV, was selected because its protoplasts showed reduced accumulation (about 10% of the CPMV yield from susceptible cowpea lines), whereas other cowpea seedlings showing susceptibility or extreme resistance to CPMV gave protoplasts supporting similar CPMV accumulation. Arlington-derived resistance has been found to be under the control of a single dominant locus, designated Cpa. Cpa-mediated resistance to CPMV is both extreme and robust. Although no virions are recovered after CPMV inoculation of Cpa cowpea, CPMV inoculation has an effect: co-inoculation of CPMV and any of several local lesion inducing challenging viruses, e.g., Cowpea severe mosaic virus (CPSMV) or Southern bean mosaic virus (SBMV), resulted in the reduction of the number of lesions produced, compared to the number of lesions produced by the challenging viruses inoculated alone, suggesting that CPMV elicits a response that reduced the accumulation of the challenging virus. This phenomenon has been termed concurrent protection. These CPMV-induced concurrent protection and eliciting activities have been mapped to the CPMV protease (24KPro, encoded by CPMV RNA1), apparently activating a resistance that is effective against not only CPMV but also any of several challenging virus including CPSMV and SBMV. Results from mutational analysis revealed that this activation seems to require the proteolytic activity of 24KPro. My research objective was to discover the cowpea protein (or proteins) that is (are) recognized and cleaved by CPMV 24KPro, using the tools of phage display. I created a phage display substrate library containing a known substrate for 24KPro and carried out the proteolytic reaction with 24KPro purified from different sources, including Nicotiana benthamiana, E. coli and a human in vitro translation Kit. To my surprise, I could not detect the cleavage of the phage display substrates despite the high theoretical sensitivity of an assay based on the detection of single bacteriophage particles. I tried other approaches of making active 24KPro, to no avail. During the course of my research, I discovered that the 24KPro gene transiently expressed on homozygous cpa/cpa cowpea, but not on Cpa/Cpa cowpea, produced detected 24KPro. When the active site cysteine residue 166 of CPMV 24KPro was substituted with an alanine residue, the mutant 24KPro accumulated in Cpa cowpea but to a lesser extent than it did in cpa cowpea, suggesting that even the inactive 24KPro may be recognized by Cpa cowpea. The possible effects of active 24KPro on Cpa/Cpa cowpea are discussed. |
