Modification of Papaya Ringspot Virus HC-Pro to Generate Effective Attenuated Mutants for Overcoming the Problem of Strain-Specific Cross Protection

Abstract

Cross protection application of HA5-1, an attenuated mutant of papaya ringspot virus (PRSV) HA strain from Hawaii, was withdrawn from Taiwan due to the narrow geographic strain specificity of HA5-1. Here, to overcome this problem, we created attenuated mutants of PRSV YK, a dominant severe strain from Taiwan, by mutating helper component protease (HC-Pro) at F 7 , R 181 , F 206 , and D 397 residues critical for potyviral pathogenicity. PRSV YK HC-Pro R 181 I, F 206 L, and D 397 N single-mutant viruses induced mild symptoms, but their adverse effects on growth of papaya plants disqualified them as useful protective viruses. However, F 7 I single-mutant and F 7 I + F 206 L double-mutant viruses displayed mild symptoms followed by recovery, and they showed a zigzag pattern of accumulation in papaya plants, indicating their potential to trigger RNA silencing and retain partial antagonistic suppression of host defense. Although F 7 I + R 181 I and F 7 I + D 397 N double-mutant viruses caused symptomless infection, they accumulated barely above mock level and, thus, were not qualified as proper protective viruses. RNA silencing suppression (RSS) analysis by agroinfiltration in Nicotiana benthamiana plants revealed that the HC-Pro F 7 I and F 7 I + F 206 L mutant proteins were weaker in RSS ability than the wild-type protein. Under greenhouse conditions, F 7 I and F 7 I + F 206 L mutant viruses were genetically stable but not aphid transmissible. Compared with the HA5-1 mutant’s low degree (10%) of protection to papaya plants, the F 7 I and F 7 I + F 206 L mutants provided complete (100%) protection to papaya and horn melon plants against strain YK. Thus, F 7 I and F 7 I + F 206 L mutants solve the problem of strain-specific protection and have great potential for control of PRSV in Taiwan.

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