Affinity purification of streptavidin using tobacco mosaic virus particles as purification tags

A new protein affinity purification system has been developed. Recombinant tobacco mosaic virus (TMV) was used as an affinity matrix for isolation and purification of the given protein of interest. In model experiments, streptavidin-specific heptapeptide sequence TLIAHPQ was inserted into TMV coat protein near the C end. This oligopeptide did not interfere significantly with viral replication, assembly, and movement. Recombinant TMV functioned as an epitope tag recognizing streptavidin in plant protein extracts. Plant protein extracts containing streptavidin were incubated with recombinant TMV virions. Affinity complexes of viral particles with the protein of interest were collected by centrifugation. Recombinant TMV-streptavidin complex was dissociated with 0.2M acetic acid, pH 4.6, and was passed through membrane filter Nanosep 300K by centrifugation. The filtrate contained pure streptavidin. Recombinant TMV was left on the filter. TMV particles collected from the filter could be used for at least two more purification cycles. The streptavidin-specific recombinant TMV system was applied successfully for purification of streptavidin from Streptomyces avidinii. The authors believe that the TMV-based affinity system can also be used for the purification of other proteins.     

Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in shape Nicotiana tabacum

We evaluated the concept for protection of plants against virus infection based on the expression of single-chain Fv (scFv) fragments in the apoplasm or cytosol of transgenic plants. Cloned cDNA of a tobacco mosaic virus (TMV)-specific scFv antibody, which binds to intact virions, was integrated into the plant expression vector pSS and used for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Xanthi-nc. Regenerated transgenic tobacco plants were analysed by northern blot, western blot and ELISA to assess expression and functionality of recombinant antibody (rAb) fragments. A significant increase of scFv levels in T₁ progeny was obtained for plants secreting apoplastic scFv antibodies but not for scFvs expressed in the cytosol. Bioassays revealed that T₁ progeny producing scFvs in different plant cell compartments showed different levels of resistance upon inoculation with TMV. The most dramatic reduction of necrotic local lesion numbers upon virus infection was observed in T₁ plants expressing scFv fragments in the cytosol. Infectivity could be reduced by more than 90%, despite the observation that protein expression levels for functional scFv antibodies were very low. Furthermore, upon inactivation of the N-resistance gene at elevated temperature, a significant portion of the T₁ progenies inhibited systemic virus spread, indicating that expression of TMV-specific cytosolic scFvs confers virus resistance in these transgenic plants. Moreover, inoculation of protoplasts isolated from transgenic and non-transgenic tobacco plants with TMV-RNA demonstrated that accumulation of virus particles is affected by cytosolic scFv expression.     

新疆辣椒轻微斑驳病毒的分离鉴定

从石河子露地辣椒病株上分离到一株病毒分离物,引起辣椒轻微斑驳。该分离物经人工接种能侵染５科９种植物。感染的辣椒种子可传毒,但桃蚜不能传播。Ｐ－２致死温度９０℃,稀释限点１０＾－９,体外保毒期５０天以上。病毒颗粒呈杆状,稍弯曲,大小为２９５±１０×１７ｎｍ;辣椒病组织超薄切片可见大量以平行或交叉排列的病毒集结体。该分离物与辣椒轻微斑驳病抗血清呈阳性反应。在辣椒上与ＴＭＶ无任何交叉保护。ＳＤＳ－聚丙烯     

Functional characterization of the mutations in Pepper mild mottle virus overcoming tomato tm‐1‐mediated resistance

In tomato plants, Pepper mild mottle virus (PMMoV) cannot replicate because the tm‐1 protein inhibits RNA replication. The resistance of tomato plants to PMMoV remains durable both in the field and under laboratory conditions. In this study, we constructed several mutant PMMoVs and analysed their abilities to replicate in tomato protoplasts and plants. We found that two mutants, PMMoV‐899R,F976Y and PMMoV‐899R,F976Y,D1098N, were able to replicate in tomato protoplasts, but only PMMoV‐899R,F976Y,D1098N was able to multiply in tomato plants. Further analysis showed that the D1098N mutation of the replication proteins weakened the inhibitory effect of the tm‐1 protein and enhanced the replication efficiency of PMMoV‐899R,F976Y,D1098N. We also observed that the infectivity of the viruses decreased in the order wild‐type PMMoV > PMMoV‐899R,F976Y > PMMoV‐899R,F976Y,D1098N in original host plants, pepper and tobacco plants. On the contrary, the single mutation D1098N abolished PMMoV replication in tobacco protoplasts. On the basis of these observations, it is likely that the deleterious side‐effects of mutations in replication proteins prevent the emergence of PMMoV mutants that can overcome tm‐1‐mediated resistance.     

Inhibition of Pepper mild mottle virus with Commercial Cellulases

Pepper mild mottle virus (PMMoV) is one of the most important pathogens of pepper crops (Capsicum annuum L.) worldwide. We have found that commercial cellulases from Trichoderma reesei and Trichoderma viride strongly inhibit PMMoV infection of plants. When purified PMMoV was mixed with the cellulases, it greatly lost the capacity to induce local lesions on leaves of Nicotiana glutinosa. Pretreatment of sweet pepper leaves (C. annuum L.) with cellulase solutions before PMMoV inoculation greatly reduced the number of infected plants. These effects were superior to the effects of known viral inhibitors such as skim milk or Lentinula edodes culture filtrates. Although the mechanism of inhibition remains unknown, this is the first report of antiviral activity ascribed to commercially available cellulases.