The experiment was performed at room temperature, and washes were done with phosphate buffer between all steps. Those escape variants that carry a C-terminal extension in the capsid protein also fail to be transmitted by nematodes. Together, these data provide structureCfunction insights into NbCGFLV recognition and the molecular mechanism leading to loss of resistance. of the family in the order and possesses a bipartite single-stranded positive-sense RNA genome (5). The structure of the icosahedral capsid is known from our previous crystal structure analysis (6) and follows Risperidone hydrochloride a pseudo= 3 triangulation. It is composed of 60 copies of the capsid protein (CP), which folds into three jelly roll sandwiches (6). Since their discovery (7), single-domain antigen-binding fragments of camelid-derived heavy chain-only antibodies, also known as nanobodies (Nbs) (8), have proven to be of outstanding interest as therapeutics against human diseases and pathogens (9C11), including viruses (12C14). Recent reports also revealed their effectiveness in conferring resistance against plant viruses. Thus, transient expression of Nbs against broad bean mottle virus attenuated the spreading of the cognate virus in (15). Recently, we showed that the constitutive expression of a single Nb (Nb23) that is specific to GFLV confers monogenic resistance to a wide range of GFLV isolates in both the model plant and grapevine (16), but the molecular basis of GFLV recognition and the mechanism of resistance induced by Nb23 are unknown. Moreover, while one of the homozygous lines tested was fully resistant to GFLV, another line showed infection at low frequency (3.2%), suggesting the existence of escape variants (EVs) containing resistance-breaking (RB) mutations susceptible to interfering with Nb23CGFLV interaction (16). To address the molecular basis of Nb23CGFLV recognition, we determined the cryo electron microscopy (cryo-EM) structure of the GFLVCNb23 complex at high JNKK1 resolution. The structure reveals that Nb23 bridges over 3 domains of the CP, and it provides Risperidone hydrochloride unprecedented insights into the epitope and the residues involved in the interface, including the mechanism of molecular recognition of the antigen-binding loops. We find a perfect correlation between mutations detected in RB variants and the Nb23 epitope observed in the structure, which explains the resistance loss in EVs. In agreement with the fact that the conformational surface epitope recognized by the Nb23 partially covers a cavity involved in vector transmission (6, 16, 17), we show that EVs preexist in natural viral populations at low frequency. We also uncover that the most frequently found EVs with extended CP are deficient in transmission by nematodes. Results To gain molecular insights into the mechanism of GFLV recognition by Nb23, precisely map the epitope, and decipher the interactions between Nb23 and the CP, we decided to analyze the structure of the GFLV viral particle decorated with Nb23. The structure of the GFLVCNb23 complex was determined by high-resolution single-particle cryo-EM, using angular reconstitution (18, 19), and refined to an average resolution of 2.8 ? (Fig. 1 and icosahedral capsid. The outer isocontour surface of the GFLVCNb23 reconstruction (Fig. 1 and and and = 3; and and and lines that constitutively express Nb23:EGFP displays various degrees of susceptibility to infection, indicating that GFLV could overcome Nb-mediated resistance (16). To further explore this partial resistance breakdown, we forced our two resistant lines toward the Risperidone hydrochloride emergence of infection events by applying high inoculum pressure (3 g vs. 300 ng of virus). Under such stringent conditions, resistance was indeed overcome by 21 d postinoculation (dpi) in 30% and 40% of plants from lines 23EG38-4 and 23EG16-9, respectively (Fig. 3 and and infected with GFLV-GHu, GFLV-CP+3, or GFLV-Tyr216His were tested by DAS-ELISA with either conventional antibodies or Nb23 for detection. Conventional antibodies recognize all GFLV isolates contrarily Risperidone hydrochloride to Nb23 that fails to detect GFLV-CP+3 and GFLV-Tyr216His, indicating reduced binding of Nb23 to GFLV EV. (transgenic lines toward GFLV GHu or GFLV-CP+3. Plants were tested by DAS-ELISA for GFLV at 21 dpi. (test; = 0.022). Number of plants tested (n) and percentage of infections (%) are provided below each.