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In plants, viral replication can be inhibited through gene silencing, which is mediated by short interfering RNA (siRNA) or microRNA (miRNA). However, under natural conditions, viruses are extremely susceptible to mutations that may decrease the efficiency of cleavage of these small RNAs (sRNAs). Therefore, a single sRNA may not provide a sufficient degree of viral resistance to transgenic plants. Potato virus Y necrotic strain (PVYN) and Potato virus Y common strain (PVYO) are the two major PVY strains that cause systemic necrosis and mottling, respectively, in tobacco. In this study, we designed specific siRNAs and miRNAs to target two regions of the PVYO replicase gene (NIb). Eight plant expression vectors containing one or two sRNAs were constructed. Luciferase activity assays showed that the designed sRNAs successfully cleaved the NIb gene of PVYO and PVYN, and the vector carrying a combined siRNA- and miRNA-based short hairpin RNA (shRNA) demonstrated the strongest inhibitory effect. These effects were confirmed through the acquisition of PVYO and PVYN resistance in transgenic sRNA-expressing Nicotiana tabacum plants. This phenomenon could be related to a plant defense mechanism in which siRNA and miRNA pathways are complementary and interact to achieve gene silencing. Furthermore, there is a tendency for the homologous small RNA sequences (PVYO) to be more effective in conferring resistance than those with imperfect homology (PVYN). Overall, these findings confirm that the use of a combined siRNA- and miRNA-based shRNAs is a promising approach for introducing viral resistance to plants through genetic engineering. [ABSTRACT FROM AUTHOR]

The storage period of European pear (Pyrus communis L.) is prolonged at low temperature, but the pear softened quickly after shifting to room temperature. However, the molecular mechanisms that influence this process are unknown. In this study, 'Docteur Jules Guyot' pears were placed at room temperature (RT) and low temperature and then turned to room temperature (LR). The fruit firmness of 'Docteur Jules Guyot' under LR decreased faster than RT after moving from low temperature. The ethylene production rate, water-soluble pectin (WSP), ionic-soluble pectin (ISP) content, and pectin degradation enzyme activity of 'Docteur Jules Guyot' under LR increased faster than RT after moving from low temperature. RNA-seq of 'Docteur Jules Guyot' pear under different storage conditions was carried out, and 1018 genes were found to be negatively correlated with fruit firmness by the weighted gene coexpression network analysis (WGCNA) method. Then, 24 structural genes and transcription factors related to fruit softening were selected to verify the RNA-Seq results. The expression of all these selected genes increased significantly after LT storage. The fruit firmness, pectin content, and pectin degradation enzyme activity of 'Laiyang' pear did not change significantly under RT and LR storage conditions. The expression of LAX2 , ARF19 , EXP1 , EXPA1 , EXP3 and PG2 was upregulated in 'Docteur Jules Guyot' pear but downregulated in 'Laiyang' pear. These genes might play important roles in the softening process of European pear. This work laid the foundation for revealing the mechanisms of European pear fruit softening. • Low temperature accelerated the softening of 'Docteur Jules Guyot' pear after shifting to room temperature. • WGCNA identifies two key modules involved in pear fruit softening. • The expression trend of some genes in European and Asian pears was completely opposite. • LAX2 , ARF19 , EXP1 , EXPA1 , EXP3 and PG2 may play important roles in European pear softening. [ABSTRACT FROM AUTHOR]