Penicillium chrysogenum polypeptide extract protects tobacco plants from tobacco mosaic virus infection through modulation of ABA biosynthesis and callose priming

Abstract

The polypeptide extract of the dry mycelium of Penicillium chrysogenum (PDMP) can protect tobacco plants from tobacco mosaic virus (TMV), although the mechanism underlying PDMP-mediated TMV resistance remains unknown. In our study, we analysed a potential mechanism via RNA sequencing (RNA-seq) and found that the abscisic acid (ABA) biosynthetic pathway and β-1,3-glucanase, a callose-degrading enzyme, might play an important role in PDMP-induced priming of resistance to TMV. To test our hypothesis, we successfully generated a Nicotiana benthamiana ABA biosynthesis mutant and evaluated the role of the ABA pathway in PDMP-induced callose deposition during resistance to TMV infection. Our results suggested that PDMP can induce callose priming to defend against TMV movement. PDMP inhibited TMV movement by increasing callose deposition around plasmodesmata, but this phenomenon did not occur in the ABA biosynthesis mutant; moreover, these effects of PDMP on callose deposition could be rescued by treatment with exogenous ABA. Our results suggested that callose deposition around plasmodesmata in wild-type plants is mainly responsible for the restriction of TMV movement during the PDMP-induced defensive response to TMV infection, and that ABA biosynthesis apparently plays a crucial role in PDMP-induced callose priming for enhancing defence against TMV.

Keywords: N. benthamiana; ABA; PDMP; callose; infection; plasmodesmata; priming; tobacco mosaic virus (TMV).

Fig. 1.

qRT–PCR-based validation of interesting DEGs (ABA2 and β-1,3-glucanase). The mRNA expression of ABA2 (A) and β-1,3-glucanase (B) were detected via qRT–PCR assay. The cycle threshold (CT) values of the target genes were normalized to the CT value of β-actin. (C) β-1,3-glucanase protein was detected by western blot assay. (D) Quantitative results of β-1,3-glucanase protein fragments. Protein relative amounts of β-1,3-glucanase were quantified relative to the expression of Rubisco. Values are means ±SD (n=6). *: P<0.05, **: P<0.01 and ***: P<0.001 versus the control treatment; ^##: P<0.001, versus TMV treatment, as determined by one-way ANOVA.

Fig. 2.

Validation of CRISPR/gRNA-mediated NbZEP genetic mutants. (A) The expression of ZEP was detected via qRT–PCR assays. The cycle threshold (CT) values of the target genes were normalized to the CT value of β-actin. (B) ABA concentration in the leaves of the NbZEP mutant and wild-type plants was determined by an ELISA. Values are means ±SD (n=20). ***: P<0.001 versus wild-type, as determined by Student’s t test.

Fig. 3.

Presence of GFP-TMV movement. (A) Movement of TMV is shown by green fluorescence under a UV lamp. Scale bar =1 cm. The green fluorescence in the inoculated leaves (B) and systemic leaves (C) was quantified via ImageJ software. Values are means ±SD (n=6). ***: P<0.001 versus TMV treatment; ^#: P<0.05 and ^###: P<0.001, as determined by one-way ANOVA. ‘m’ indicates ABA biosynthesis mutant.

Fig. 4.

Presence of callose fluorescence and callose-related protein (β-1,3-glucanase). (A) Callose deposition is shown as turquoise fluorescence after staining with aniline blue and imaged under an ultraviolet excitation filter (BP 330–385 nm) via a fluorescence microscope. Scale bar =100 µ m. (B) Relative callose intensities emitted by aniline blue-stained callose deposits (n=6). The turquoise fluorescence was regarded as the regions of interest (ROI). The turquoise fluorescence was chosen and quantified manually using ImageJ software. (C) Western blot validation of β-1,3-glucanase expression. (D) Protein fragments were quantified relative to the expression of Rubisco via ImageJ software (n=3). Values are means ±SD. *: P<0.05 and ***: P<0.001 versus TMV treatment; ^#: P<0.05 and ^###: P<0.001, as determined by one-way ANOVA. ‘m’ indicates ABA biosynthesis mutant.

Fig. 5.

Analysis of immunocolloidal gold coupled with transmission electron microscopy of callose deposits around plasmodesmata. (A) β -1,3-glucan and β -1,3-glucanase were detected around plasmodesmata in the different treatments. Scale bar =200 nm. Quantitative results of β -1,3-glucan (B) and β -1,3-glucanase (C) gold particles around plasmodesmata. The number of gold particles was counted per individual plasmodesma (8–10 plasmodesmata were analysed per sample). Values are means ± SD (n=3). ***: P<0.001 versus TMV treatment; ^#: P<0.05, ^##: P<0.01 and ^###: P<0.001, as determined by one-way ANOVA. PD: plasmodesma. The red arrows indicate callose particles. ‘m’ indicates ABA biosynthesis mutant.

Fig. 6.

Plasmodesmata structural parameters. (A) Plasmodesmata ultrastructure was observed via transmission electron microscopy. (B) Plasmodesmata diameters (8–10 plasmodesma were analysed per sample) were measured at the neck region via the TEM Imaging & Analysis software. Scale bar =200 nm. Values are means ±SD (n=27). *: P<0.05 and ***: P<0.001 versus TMV treatment; ^##: P<0.01, as determined by one-way ANOVA. The red arrows show the neck of plasmodesmata. The letter ‘m’ indicates ABA biosynthesis mutant.