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A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants. Image 1 • A scFv library previously screened in a Y2H system against a human rotavirus was tested against CMV 2a protein derivatives. • Positive scFvs were analyzed for their interaction with CMV 2a protein derivatives in mammalian transient expression system. • Tobacco plants transformed with four selected scFvs had reduced levels of CMV and showed premature recovery from infection. [ABSTRACT FROM AUTHOR]

Temperature is an important environmental factor controlling plant growth, development, and immune response. However, the role of temperature in plant disease resistance is still elusive. In the present study, the potential effects of temperature on the interaction between Nicotiana tabacum and Cucumber mosaic virus (CMV) were investigated. Our results indicated that N. tabacum plants displayed severe symptoms at early stage of post inoculation at high temperature (HT, 28°C), associated with higher viral replication level, more serious stress damages. By contrast, low temperature (LT, 18°C) effectively delayed the replication of CMV compared with elevated temperatures. Additionally, quantitative real-time PCR analyses revealed that lower temperatures (≤ 24°C) promote salicylic acid (SA) dependent responses, whereas higher temperatures (> 24°C) activate the genes expression of jasmonic acid (JA) pathway. Interestingly, the dark green islands (DGIs) appeared much earlier in CMV-inoculated plants grown at HT compared with those at LT and the accumulation of virus small interfering RNAs in plants were significantly up-regulated under elevated temperatures at early stage of post inoculation. Taken together, these results indicated that temperature changes had important effects on plant defence response, and different temperatures could induce different immune pathways of N. tabacum against CMV infection. [ABSTRACT FROM AUTHOR]

Light is an important environment factor controlling plant growth, development, and nutritional quality and is also one of the most important factors inducing plant defence. In this study, we assayed the potential effects of light quality on the interaction between Nicotiana tabacum and cucumber mosaic virus ( CMV). Our results indicated that white light-treated N. tabacum plants displayed obvious symptoms at early stage postinoculation, while the symptoms were significantly inhibited under red light and blue light. Western blotting and quantitative real-time PCR ( qRT- PCR) analyses showed that blue light and red light can effectively delay the replication of CMV compared with white light. The activities of various reactive oxygen species ( ROS)-scavenging enzymes and reducing substances [reduced glutathione ( GSH) and ascorbic acid ( ASA)] were increased under blue light and red light. In addition, hormone measurements and qRT- PCR analyses revealed that salicylic acid ( SA)-mediated signalling pathway plays positive role in the related regulation, and cytokinin ( CTK) may also participate in them. Furthermore, we found that the formation of dark green islands ( DGIs) was significantly suppressed in plants under red light and blue light at 30 days postinoculation (Dpi). However, the accumulation of virus in plants under different light conditions had no notable differences at later stage of postinoculation. Taken together, these results indicated that red light and blue light could effectively delay symptom expression and replication of CMV on N. tabacum at the relatively earlier stage postinoculation. [ABSTRACT FROM AUTHOR]

Disease development in Nicotiana tabacum after inoculation with cucumber mosaic virus (CMV) was accelerated when plants were grown in high light (300 µmol m s, HL) compared with those grown in low light (70 µmol m s, LL). Several characteristic defense reactions, including release of reactive oxygen species, accumulation of salicylic acid (SA), expression of SA-mediated pathogenesis related (PR) proteins and the systemic development of disease, were proved to be light dependent. Plants exposed to HL or medium light (150 µmol m s, ML) had serious oxidative damage caused by CMV infection compared with LL-plants. In addition, the levels of SA and mRNA transcripts for non- expressor of pathogenesis- related gene 1 in CMV-inoculated plants were the highest under LL with decreasing photon flux density. In HL-exposed plants, the expression of PR1 and PR2 were repressed, but the expression of PR5 was not significantly altered, revealing a complex cross talk between light intensity and the SA signaling pathway in regulating N. tabacum response to the infection of CMV. Taken together, our results revealed that increased light intensity reduced the resistance ability of N. tabacum to CMV, correlating with repression of the SA-dependent signaling pathway and a change in the cellular redox status in plants cells. [ABSTRACT FROM AUTHOR]

Key message: There are significant differences between the DGIs and LGTs. Additionally, most of the characteristics indicate that the DGIs are more similar to recovered tissue and can resist viral attacks. Abstract: Dark green islands (DGIs) surrounded by light green tissues (LGTs) are common leaf symptoms of plants that are systemically infected by various mosaic viruses. We performed cytological, physiological and molecular biological analyses of the DGIs and LGTs in cucumber mosaic virus-infected Nicotiana tabacum leaves. Our results indicated that the DGIs contained less virus than did the LGTs. Compared to the LGTs, the DGIs contained higher levels of the metabolites involved in plant defence. The contents of reduced glutathione and ascorbic acid were increased in the DGIs to reach levels that were even higher than those of control plants. Moreover, hormone measurements and quantitative real-time PCR analysis revealed that the endogenous salicylic acid, ethylene and defence genes mediated these elevations by playing positive roles in the regulation of the DGIs responses to viral infection. The accumulation of cytokinin was also much greater in the DGIs than in the LGTs. Finally, northern blotting analysis indicated that the accumulation of viral small interfering RNAs was decreased in the DGIs compared to the LGTs. Taken together, these results suggest that DGIs might represent leaf areas that have recovered from viral infection due to locally enhanced defence responses. [ABSTRACT FROM AUTHOR]