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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]

Key message: Soybean expressing theCucumber mosaic virus 2b gene manifests seed coat pigmentation due to suppression of endogenous RNA silencing but no other morphological abnormality. This gene may help prevent transgene silencing.RNA silencing is an important mechanism for gene regulation and antiviral defense in plants. It is also responsible for transgene silencing, however, and thus hinders the establishment of transgenic plants. The 2b protein of Cucumber mosaic virus (CMV) functions as a suppressor of RNA silencing and therefore might prove beneficial for stabilization of transgene expression. We have now generated transgenic soybean that harbors the 2b gene of a CMV-soybean strain under the control of a constitutive promoter to investigate the effects of 2b expression. No growth abnormality was apparent in 2b transgenic plants, although the seed coat was pigmented in several of the transgenic lines. Genes for chalcone synthase (CHS), a key enzyme of the flavonoid pathway, are posttranscriptionally silenced by the inhibitor ( I) locus in nonpigmented (yellow) soybean seeds. The levels of CHS mRNA and CHS small interfering RNA in strongly pigmented 2b transgenic seed coats were higher and lower, respectively, than those in the seed coat of a control transgenic line. The expression level of 2b also correlated with the extent of seed coat pigmentation. On the other hand, introduction of the 2b gene together with the DsRed2 gene into somatic embryos prevented the time-dependent decrease in transient DsRed2 expression. Our results indicate that the 2b gene alone is able to suppress RNA silencing of endogenous CHS genes regulated by the I locus, and that 2b is of potential utility for stabilization of transgene expression in soybean without detrimental effects other than seed coat pigmentation. [ABSTRACT FROM AUTHOR]

Key message: Soybean expressing theCucumber mosaic virus 2b gene manifests seed coat pigmentation due to suppression of endogenous RNA silencing but no other morphological abnormality. This gene may help prevent transgene silencing.RNA silencing is an important mechanism for gene regulation and antiviral defense in plants. It is also responsible for transgene silencing, however, and thus hinders the establishment of transgenic plants. The 2b protein of Cucumber mosaic virus (CMV) functions as a suppressor of RNA silencing and therefore might prove beneficial for stabilization of transgene expression. We have now generated transgenic soybean that harbors the 2b gene of a CMV-soybean strain under the control of a constitutive promoter to investigate the effects of 2b expression. No growth abnormality was apparent in 2b transgenic plants, although the seed coat was pigmented in several of the transgenic lines. Genes for chalcone synthase (CHS), a key enzyme of the flavonoid pathway, are posttranscriptionally silenced by the inhibitor ( I) locus in nonpigmented (yellow) soybean seeds. The levels of CHS mRNA and CHS small interfering RNA in strongly pigmented 2b transgenic seed coats were higher and lower, respectively, than those in the seed coat of a control transgenic line. The expression level of 2b also correlated with the extent of seed coat pigmentation. On the other hand, introduction of the 2b gene together with the DsRed2 gene into somatic embryos prevented the time-dependent decrease in transient DsRed2 expression. Our results indicate that the 2b gene alone is able to suppress RNA silencing of endogenous CHS genes regulated by the I locus, and that 2b is of potential utility for stabilization of transgene expression in soybean without detrimental effects other than seed coat pigmentation. [ABSTRACT FROM AUTHOR]