A strain-specific segment of the RNA-dependent RNA polymerase of grapevine fanleaf virus determines symptoms in Nicotiana species

Abstract

Factors involved in symptom expression of viruses from the genus Nepovirus in the family Secoviridae such as grapevine fanleaf virus (GFLV) are poorly characterized. To identify symptom determinants encoded by GFLV, infectious cDNA clones of RNA1 and RNA2 of strain GHu were developed and used alongside existing infectious cDNA clones of strain F13 in a reverse genetics approach. In vitro transcripts of homologous combinations of RNA1 and RNA2 induced systemic infection in Nicotiana benthamiana and Nicotiana clevelandii with identical phenotypes to WT virus strains, i.e. vein clearing and chlorotic spots on N. benthamiana and N. clevelandii for GHu, respectively, and lack of symptoms on both hosts for F13. The use of assorted transcripts mapped symptom determinants on RNA1 of GFLV strain GHu, in particular within the distal 408 nt of the RNA-dependent RNA polymerase (1E Pol ), as shown by RNA1 transcripts for which coding regions or fragments derived thereof were swapped. Semi-quantitative analyses indicated no significant differences in virus titre between symptomatic and asymptomatic plants infected with various recombinants. Also, unlike the nepovirus tomato ringspot virus, no apparent proteolytic cleavage of GFLV protein 1E Pol was detected upon virus infection or transient expression in N. benthamiana. In addition, GFLV protein 1E Pol failed to suppress silencing of EGFP in transgenic N. benthamiana expressing EGFP or to enhance GFP expression in patch assays in WT N. benthamiana. Together, our results suggest the existence of strain-specific functional domains, including a symptom determinant module, on the RNA-dependent RNA polymerase of GFLV.

Allopolyploidy and the evolution of plant virus resistance

“…The only instances of visible symptoms were for GFLV-GHu on N. benthamiana, N. clevelandii, N. goodspeedii and 2 × ( N. tabacum × N. benthamiana ), and for ToRSV-AP on N. benthamiana and 2 × ( N. tabacum × N. benthamiana ). GFLV-GHu symptoms on N. benthamiana and N. clevelandii were consistent with those previously described [47], and included vein clearing on N. benthamiana and amorphous ring-like mottling on N. clevelandii . GFLV-GHu symptoms on N. goodspeedii included vein clearing analogous to that observed for N. benthamiana .…”

“…Plants were designated as susceptible (category 6) when 100% of the plants became infected and virus was present in all tested leaves. N. benthamiana and N. clevelandii were susceptible to GFLV-F13 and GFLV-GHu, as expected [47], N. goodspeedii and N. attenuata were susceptible to GFLV-GHu, and N. benthamiana and 4 × ( N. quadrivalvis × N. tabacum ) were susceptible to ToRSV-AP (Figure 1; Table 2). …”

“…The observation of ringspot symptoms on the GFLV-GHu-inoculated 2 × ( N. tabacum x N. benthamiana ) amphihaploid suggests that the vein clearing symptomology typical of N. benthamiana infection [47] is a recessive trait. Similarly, while ToRSV-AP produced necrosis on N. benthamiana , necrosis was not observed on the 2 × ( N. tabacum × N. benthamiana ) amphihaploid or on N. tabacum .…”

“…GFLV strain F13 from France [72,73] and strain GHu from Hungary [47,74] were isolated from infected grapevines, and ToRSV strain AP was isolated from an infected apricot tree in New York State [75]. GFLV and ToRSV strains were maintained in N. benthamiana .…”

Nanobody‐mediated resistance to Grapevine fanleaf virus in plants

“…To assess the effect of Nb23:EGFP on GFLV in vivo , we produced transgenic N. benthamiana constitutively expressing Nb23:EGFP or EGFP only as a control. Initially, segregating T1 plants were challenged with GFLV isolate GHu because of its capacity to induce symptoms on N. benthamiana (Vigne et al ., ). As expected, typical infection symptoms were observed in nearly 100% of the plants from the 10 independent lines constitutively expressing EGFP.…”