Tobacco EXPA4 plays a role in Nicotiana benthamiana defence against virus attack and affects antioxidative metabolism and phytohormone-mediated immunity responses in tobacco. Expansins are cell wall-loosening proteins known for their endogenous functions in cell wall extensibility during plant growth. The effects of expansins on plant growth, developmental processes and environment stress responses have been well studied. However, the exploration of expansins in plant virus resistance is rarely reported. In the present study, virus-induced gene silencing (VIGS) and Agrobacterium-mediated transient overexpression were conducted to investigate the role of Nicotiana tabacum alpha-expansin 4 (EXPA4) in modulating Tobacco mosaic virus (TMV-GFP) resistance in Nicotiana benthamiana. The results indicated that silencing of EXPA4 reduced the sensitivity of N. benthamiana to TMV-GFP, and EXPA4 overexpression accelerated virus reproduction on tobacco. In addition, our data suggested that the changes of virus accumulation in response to EXPA4 expression levels could further affect the antioxidative metabolism and phytohormone-related pathways in tobacco induced by virus inoculation. EXPA4-silenced plants with TMV-GFP have enhanced antioxidant enzymes activities, which were down-regulated in virus-inoculated 35S:EXPA4 plants. Salicylic acid accumulation and SA-mediated defence genes induced by TMV-GFP were up-regulated in EXPA4-silenced plants, but depressed in 35S:EXPA4 plants. Furthermore, a VIGS approach was used in combination with exogenous phytohormone treatments, suggesting that EXPA4 has different responses to different phytohormones. Taken together, these results suggested that EXPA4 plays a role in tobacco defence against viral pathogens.
Since they function as cell wall-loosening proteins, expansins can affect plant growth, developmental processes and environmental stress responses. Our previous study demonstrated that changes in Nicotiana tabacum α-expansin 4 (EXPA4) expression affect the sensitivity of tobacco to Tobacco mosaic virus [recombinant TMV encoding green fluorescent protein (TMV-GFP)] infection by Agrobacterium-mediated transient expression. In this study, to characterize the function of tobacco EXPA4 further, EXPA4 RNA interfernce (RNAi) mutants and overexpression lines were generated and assayed for their tolerance to abiotic stress and resistance to pathogens. First, the differential phenotypes and histomorphology of transgenic plants with altered EXPA4 expression indicated that EXPA4 is essential for normal tobacco growth and development. By utilizing tobacco EXPA4 mutants with abiotic stress, it was demonstrated that RNAi mutants have increased hypersensitivity to salt and drought stress. In contrast, the overexpression of EXPA4 in tobacco conferred greater tolerance to salt and drought stress, as indicated by less cell damage, higher fresh weight, higher soluble sugar and proline accumulation, and higher expression levels of several stress-responsive genes. In addition, the overexpression lines were more susceptible to the viral pathogen TMV-GFP when compared with the wild type or RNAi mutants. The induction of the antioxidant system, several defense-associated phytohormones and gene expression was down-regulated in overexpression lines but up-regulated in RNAi mutants when compared with the wild type following TMV-GFP infection. In addition, EXPA4 overexpression also accelerated the disease development of Pseudomonas syringae DC3000 on tobacco. Taken together, these results suggested that EXPA4 appears to be important in tobacco growth and responses to abiotic and biotic stress.
Dark green islands (DGIs) surrounded by light green tissues (LGTs) are common leaf symptoms of plants that are systemically infected by various viruses that induce leaf mosaic in infected plants. The inoculation of Cucumber mosaic virus (CMV) in Nicotiana tabacum produced a commonly occurring sequence of classic patterns of DGIs and LGTs. Previous studies confirmed that there are significant differences between DGIs and LGTs in terms of physiology, biochemistry and molecular biology, but the mechanisms by which DGIs form remain unclear. To investigate the global gene expression changes that occur in these special tissues, individual differential gene expression tag libraries were constructed from three total RNA samples isolated from DGIs, LGTs and control plants (CK) and were sequenced using an Illumina HiSeqTM 2000. An analysis of differentially expressed genes (DEGs) and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. These analyses revealed the differences between DGIs, LGTs and CK. GO enrichment and KEGG pathway analyses suggested that several pathways related to photosynthesis and chlorophyll metabolism were enriched in DGIs compared to LGTs and CK. Several pathways related to apoptosis were significantly up-regulated in LGTs compared to DGIs. Additionally, we identified sets of DEGs that may be related to the formation or development of DGIs and LGTs. Our systematic analyses provide comprehensive transcriptomic information regarding DGIs and LGTs in CMV-infected N. tabacum. These data will help characterize the detailed mechanisms of DGI and LGT formation.
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