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?2). Interestingly, when mutation 48W was introduced into the infectious clone of isolate CIa, the mutated clone infected neither resistant nor susceptible plants. Then, mutation 48W fixed in three isolates Angiogenesis inhibitor of strain S1-ca and in one isolate of the S2�CS3 strain (isolate Ma203) was lethal in the infectious clone CIa of the S2�CS3 strain. The rymv1�C3 resistance was overcome after direct inoculation of transcripts of the avirulent infectious clone; systemic symptoms developed and the mutation 41P was fixed. This is the first report of adaptation of an infectious clone to the rymv1 resistance. In avirulent isolates, RB mutations at codons 41 and 52 emerged at a similar frequency. A first mutation either at codon 41 or 52��there was no order of appearance��was often followed by the fixation of the other one, as revealed when following the course of infection ( Fig.?3). Consistently, mutation 52Y was fixed after inoculation of the infectious clone mutated with 41P. In no instance was a mutant displaced by the alternate one during the rymv1�C3 RB process. The quantitative RT-PCR technique (qRT-PCR) was applied to assess the viral accumulation in rymv1�C3 resistant Enzalutamide supplier plants after inoculation of the avirulent isolate, of the single RB mutants, and of the double RB mutants. The following trends were apparent ( Fig.?4). After inoculation of the avirulent isolate, the sensitive qRT-PCR technique detected a low but consistent viral RNA accumulation, above the threshold level set with test of non-inoculated leaves. When a single RB mutation was fixed, be it 52Y, 41P or 41A, the virus accumulation of the single mutant was significantly higher than the avirulent isolate (P?=?0.003 in one-sided Student test with unequal variance), i.e. 10 to 102-fold greater than the WT. When both mutations were fixed together, be it 41P and 52Y or 41A and 52Y, the virus accumulation of the double mutants was significantly higher than the single mutants (P?=?0.002 in one-sided Student test with unequal variance), i.e. c. 105 greater than the avirulent isolate. After this stepwise RB accumulation, the virus content in resistant plants reached c. 1012 viral RNA copies per milligram of leaf. The 21 isolates which broke rymv1�C3 resistance had a threonine at codon 49 of the VPg ( Fig.?1, Dimethyl sulfoxide Table?1). By contrast, none of the 49 isolates with a glutamic acid at this position became virulent when inoculated to rymv1�C3. Increasing the number of plants challenged with isolates with a glutamic acid did not result in a successful infection. The link between the ability to break rymv1�C3 resistance and the amino-acid at codon 49 was highly significant (��2?=?37.9, P?