Cross Protection Of The Engineered Mild Mutants To Tobacco Vein Banding Mosaic Virus

Cross protection is a phenomenon that prior infection with one (protecting) plant viruswill prevent or interfere with superinfection by another challenging virus, usually relatedvirus. Cross-protection has been successfully used to control plant viral diseases induced bytomato mosaic virus, citrus tristeza virus, papaya ring spot virus, cucumber mosaic virus andzucchini yellow mosaic virus. Although several attenuated viruses have been used in diseasecontrol in commercial fields, the practical use of cross protection is still in debate. The use ofa mild strain for protection has some disadvantages. For examples, the mild strains maymutate to severe strains or spread to non-target crops, and they usually are only effective toclosely related strains, and they may have synergism with other co-infecting viruses and causemore severe losses.In the present study, multiple mutations were introduced to the infectious clone oftobacco vein banding mosaic virus (TVBMV; genus Potyvirus), by which mild strain ofTVBMV with less virulence and high safety were obtained. The main results read as follows:1. Construction of mild TVBMV strains with two or more mutations. The mutations ofRITC to EITC (A) which abolished aphid transmission, NRT to ART (B), FRNK to FINK (C),CDN to CKN (D), and IQN to DEN (E) that reduced the virulence were introduced to thehelper component-proteinase (HC-Pro) encoding region in TVBMV to generate novel mildstrains. Here we obtained four mild strains with double mutations (BC, BD, CE, DE), fourwith triple mutations (ACE, ADE, BCE, BDE), and two with four mutations (ABCE, ABDE).When inoculated to Nicotiana benthamiana using Agrobacterium tumefeciens GV3101,wild type TVBMV (pCamTVBMV-GFP) induced severe symptom, with yellow mosaic,distortion and epinasty, and strong green fluorescence was observed on the systemic leaves at5-7days post infiltration (dpi). However, no apparent symptom was observed wheninoculated with the ten mutants. Weak or invisible fluorescence was observed in the systemicleaves. ELISA readings revealed that the CP accumulation of the mutant viruses was low andfar less than that of the wild-type virus. The results of RT-PCR indicated that viral RNA canbe detected from the systemic leaves, indicating that the mutants can replicate and movesuccessfully. The mutants with two or more mutations induced can systemically infect plantsof Nicotiana tabacum but induced no apparent symptom, either.2. Plants of N. benthamiana were inoculated by the ten mild mutants and wild typePVX (V/V=1/1) using A. tumefeciens. The plants inoculated with wild type TVBMV andPVX showed severe systemic mosaic symptom, and necrosis in the systemic leaves. But plants of N. benthamiana inoculated with the mild mutants and PVX just appeared systemicmosaic symptoms similar to that induced by wild type PVX or even slighter. ELISA readingsrevealed that the accumulations of PVX CP were lower than that of the wild-type PVX. Theseresults indicated that the mild mutants had no synergistic effects with wild type virus (PVX).Same conclusion can be drawn when inculated to plants of N. tabacum.3. Some TVBMV mutants abolished aphid transmission ability. Plants of N. benthamianawere inoculated by the four mild mutants ACE, ADE, ABCE and ABDE and wild typeTVBMV. After20days, virus-free aphids were fasted for2h and allowed to feed oninoculated plants for5min, transferred to the healthy N. benthamiana plants, and then killedby spraying agrochemicals12h later. After6days, N. benthamiana plants inoculated withaphids feeding wild type virus showed no apparent symptom but, under ultraviolet light,showed green fluorescence on some region of inoculated leaves. After8days, those plantsshowed distinct vein clearing and, under ultraviolet light, green fluorescence on systemicleaves. However, N. benthamiana plants inoculated with aphids feeding mild mutants ACE,ADE, ABCE and ABDE showed no distinct symptoms under normal light, no greenfluorescence under ultraviolet light, even eight days after inoculation. These results suggestedthat mild mutants ACE, ADE, ABCE and ABDE lost aphid transmission ability.4. The mutants provided good cross protection to wild-type TVBMV. The N. benthamianawere inoculated by the mild mutants as the protective inoculation. At3,7and11days afterthe protective inoculation, the first fully expanded leaves of the plants were mechanicallychallenged with wild type TVBMV-GFP. The titers of the mutants in infected N.benthamiana plants were monitored at5,10and15dpi by ELISA. The results showed thatthe symptoms, fluorescence intensity and virus accumulation in protected N. benthamianaplants were reduced; and better protection can be achieved if the plants are protected11daysbefore chanllenge inoculation. The mutant B can provide better protection than other mutants.