Molecular Regulation of Host Defense Responses Mediated by Biological Anti-TMV Agent Ningnanmycin

Abstract

Ningnanmycin (NNM) belongs to microbial pesticides that display comprehensive antiviral activity against plant viruses. NNM treatment has been shown to efficiently delay or suppress the disease symptoms caused by tobacco mosaic virus (TMV) infection in local-inoculated or systemic-uninoculated tobacco leaves, respectively. However, the underlying molecular mechanism of NNM-mediated antiviral activity remains to be further elucidated. In this study, 414 differentially expressed genes (DEGs), including 383 which were up-regulated and 31 down-regulated, caused by NNM treatment in TMV-infected BY-2 protoplasts, were discovered by RNA-seq. In addition, KEGG analysis indicated significant enrichment of DEGs in the plant-pathogen interaction and MAPK signaling pathway. The up-regulated expression of crucial DEGs, including defense-responsive genes, such as the receptor-like kinase FLS2, RLK1, and the mitogen-activated protein kinase kinase kinase MAPKKK, calcium signaling genes, such as the calcium-binding protein CML19, as well as phytohormone responsive genes, such as the WRKY transcription factors WRKY40 and WRKY70, were confirmed by RT-qPCR. These findings provided valuable insights into the antiviral mechanisms of NNM, which indicated that the agent induces tobacco systemic resistance against TMV via activating multiple plant defense signaling pathways.

Keywords: BY-2 protoplasts; Ningnanmycin; antiviral agents; resistance genes; transcriptome analysis.

Figure 1

Effects of Ningnanmycin (NNM) treatment on tobacco mosaic virus (TMV)-inoculated Nicotiana benthamiana and BY-2 protoplasts. (A) Schematic representation of TMV-inoculated leaves (I) and the un-inoculated upper leaves (U) of N. benthamiana with the treatment of distilled H₂O, 100 μg/mL and 50 μg/mL NNM at 7 dpi. (B) Northern blot analysis of the accumulation of TMV RNA from the inoculated leaves and upper leaves and relative accumulation of genomic RNA (gRNA) are shown. The ribosomal RNA (rRNA) is shown below the northern blots as a loading control. (C) Schematic representation of NNM treatment on TMV-inoculated BY-2 protoplasts. (D) RNA accumulations of TMV in BY-2 protoplasts under various concentrations of NNM treatment and relative accumulation of positive- and negative-strand gRNA are shown.

Figure 2

NNM-induced differential gene expression in BY-2 protoplasts. (A) Numbers of up-regulated genes (in red) and down-regulated differentially expressed genes (in blue) were shown (false discovery rate (FDR) < 0.05 and ≥ 2-fold change). Black dots indicate non-differentially expressed genes (FDR ≥ 0.05). (B) Differentially expressed genes (DEGs)—383 up-regulated, and 31 down-regulated—are listed. (C) RT-qPCR verification on regulation of nine DEGs induced by NNM. An equal volume of sterile water treatment was used as a control. * indicate a significant difference (p < 0.05) and ** indicate a significant difference (p < 0.01).

Figure 3

DEGs enriched in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis induced by NNM. (A) GO terms of three categories significantly enriched in DEGs of NNM vs. CK involved in molecular function, cellular component, and biological process. (B) KEGG pathways of the significantly enriched DEGs. The rich factor reflects the degree of enriched DEGs in a given pathway. The number of enriched DEGs in the pathway is indicated by the circle area, and the circle color represents the ranges of the corrected p-value.

Figure 4

A model for crucial genes regulation involved in anti-TMV mechanisms by treatment of NNM.