Silencing of a Nicotiana benthamiana ascorbate oxidase gene reveals its involvement in resistance against cucumber mosaic virus.

MAIN CONCLUSION: Silencing of an ascorbate oxidase (AO) gene in N. benthamiana enhanced disease severity from cucumber mosaic virus (CMV), showing higher accumulation and expansion of the spreading area of CMV. A Nicotiana benthamiana ascorbate oxidase (NbAO) gene was found to be induced upon cucumber mosaic virus (CMV) infection. Virus-induced gene silencing (VIGS) was employed to elucidate the function of AO in N. benthamiana. The tobacco rattle virus (TRV)-mediated VIGS resulted in an efficient silencing of the NbAO gene, i.e., 97.5% and 78.8% in relative quantification as compared to the control groups (TRV::eGFP- and the mock-inoculated plants), respectively. In addition, AO enzymatic activity decreased in the TRV::NtAO-silenced plants as compared to control. TRV::NtAO-mediated NbAO silencing induced a greater reduction in plant height by 15.2% upon CMV infection. CMV titer at 3 dpi was increased in the systemic leaves of NbAO-silenced plants (a 35-fold change difference as compared to the TRV::eGFP-treated group). Interestingly, CMV and TRV titers vary in different parts of systemically infected N. benthamiana leaves. In TRV::eGFP-treated plants, CMV accumulated only at the top half of the leaf, whereas the bottom half of the leaf was "occupied" by TRV. In contrast, in the NbAO-silenced plants, CMV accumulated in both the top and the bottom half of the leaf, suggesting that the silencing of the NbAO gene resulted in the expansion of the spreading area of CMV. Our data suggest that the AO gene might function as a resistant factor against CMV infection in N. benthamiana.

Influence of root-knot nematode infestation on antioxidant enzymes, chlorophyll content and growth in Pogostemon cablin (Blanco) Benth.

Plants adapt themselves to overcome adverse environmental conditions, and this involves a plethora of concurrent cellular activities. Physiological experiments or metabolic profiling can quantify this response. Among several diseases of Pogostemon cablin (Blanco) Benth. (Patchouli), root-knot nematode infection caused by Meloidogyne incognita (Kofoid and White) Chitwood causes severe damage to the plant and hence, the oil production. In the present study, we identified M. incognita morphologically and at molecular level using sequenced characterized amplified region marker (SCAR). M. incognita was artificially inoculated at different levels of second stage juveniles (J2) to examine the effect on Patchouli plant growth parameters. Peroxidase and polyphenol oxidase enzyme activity and changes in the total phenol and chlorophyll contents in M. incognita was also evaluated in response to infection. The results have demonstrated that nematode infestation leads to increased peroxidase activities in the leaves of the patchouli plants and thereby, increase in phenolic content as a means of defence against nematode infestation. Chlorophyll content was also found decreased but no changes in polyphenol oxidase enzyme activity.

Comparative transcriptome profiling reveals differential defense responses among Alternaria brassicicola resistant Sinapis alba and susceptible Brassica rapa.

Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba, a close relative of Brassicas, and the highly susceptible Brassica rapa. The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa. Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola, which will aid in devising strategies for breeding resistant varieties of oilseed Brassica.