Targeting of the Hybrid Bamboo BDDnaJ by Pathogen Effector ApcE12 Regulates the Unfolded Protein Response

Abstract

The shoot blight disease of Bambusa pervariabilis × Dendrocalamopsis grandis, caused by Arthrinium phaeospermum, threatens bamboo's ecological and economic value. This study explores the pathogenic effector ApcE12's role in modulating plant immunity through interactions with the host proteins BDClp and BDDnaJ. ApcE12 directly interacts with BDDnaJ, a vital regulator of the unfolded protein response (UPR), as validated through yeast two-hybrid, bimolecular fluorescence complementation, and GST pull-down assays. Functional analyses demonstrated that silencing BDDnaJ reduces UPR, activating programmed cell death (PCD) and blocking further pathogen infection to enhance plant resistance. BDDnaJ was found to regulate BDBiP protein stability by interacting with BDBiP, and it is this mechanism by which the pathogenic effector ApcE12 regulates BDDnaJ expression, enhances UPR signalling, and inhibits PCD, thereby promoting infection. These findings deepen our understanding of how fungal effectors manipulate UPR and PCD to overcome plant defences, providing novel insights for developing resistance strategies in bamboo species.

Keywords: Arthrinium phaeospermum; Bambusa pervariabilis × Dendrocalamopsis grandis; programmed cell death; protein–protein interactions; unfolded protein response.

FIGURE 1

Interaction between ApcE12 and BDClp, ApcE12, and BDDnaJ. (a) One‐on‐one yeast two‐hybrid assay results of ApcE12 with BDClp (top) and ApcE12 with BDDnaJ (bottom). (b) Bimolecular fluorescence complementation results showing the interaction between the effector protein ApcE12 and the interacting proteins Clp and DnaJ in Nicotiana benthamiana. YCE(M) is the empty vector of pSPYCE(M) as a control. (c) Glutathione S‐transferase (GST) pull‐down results of target proteins Clp and DnaJ.

FIGURE 2

Analysis of resistance function of BDDnaJ gene. (a) Expression patterns of BDClp and BDDnaJ genes in Bambusa pervariabilis × Dendrocalamopsis grandis before and after inoculation with Arthrinium phaeospermum. (b) Relative expression levels of BDClp and BDDnaJ in BDClp‐ and BDDnaJ‐overexpressing transgenic plants and silenced transgenic plants compared to the wild type (WT). (c) Symptoms of whole plants of wild‐type and transgenic plants 28 days after pathogen inoculation. (d) Local symptoms of stem segments and leaves of wild‐type and transgenic plants 28 days after pathogen inoculation. (e) Disease index of wild‐type and transgenic plants from 0 to 28 days (dpi) after pathogen inoculation. (f) Relative expression levels of BDClp and BDDnaJ genes in wild‐type and transgenic plants 28 days after pathogen inoculation. * and different lowercase letters indicate statistically significant differences after the Waller–Duncan test (p < 0.05). Silenced: ΔBDDnaJ‐RNAi, ΔBDClp‐RNAi; overexpression: ΔBDDnaJ‐OE, ΔBDClp‐OE.

FIGURE 3

Analysis of physiological indexes regulated by BDDnaJ in Bambusa pervariabilis × Dendrocalamopsis grandis. (a) Catalase (CAT) activity in wild‐type and BDClp‐ and BDDnaJ‐overexpressing and silenced transgenic plants after inoculation with Arthrinium phaeospermum. Days post‐inoculation, dpi. (b) Superoxide dismutase (SOD) activity in wild‐type and BDClp‐ and BDDnaJ‐overexpressing and silenced transgenic plants. (c) Hydrogen peroxide (H₂O₂) content in wild‐type and BDClp‐ and BDDnaJ‐overexpressing and silenced transgenic plants. (d) Chlorophyll content in wild‐type and BDClp‐ and BDDnaJ‐overexpressing and silenced transgenic plants. (e) Jasmonic acid (JA) content in wild‐type and BDClp‐ and BDDnaJ‐overexpressing and silenced transgenic plants. (f) Salicylic acid (SA) content in wild‐type and BDClp‐ and BDDnaJ‐overexpressing and silenced transgenic plants. Different lowercase letters denote statistically significant differences based on the Waller–Duncan test (α = 0.05). Silenced: ΔBDDnaJ‐RNAi, ΔBDClp‐RNAi; overexpression: ΔBDDnaJ‐OE, ΔBDClp‐OE.

FIGURE 4

BDClp and BDDnaJ localised to the endoplasmic reticulum. mCherry‐HDEL is an endoplasmic reticulum localisation signal marker. BDDnaJ‐eGFP and BDClp‐eGFP fusion proteins (green) co‐localise in endoplasmic reticulum signalling (red). The second row of images is the centre of the field of view of the first row of images.

FIGURE 5

BDClp cooperates with BDDnaJ in conferring resistance in Bambusa pervariabilis × Dendrocalamopsis grandis to Arthrinium phaeospermum. (a) Whole plant symptoms of ΔBDDnaJ‐OE and ΔBDClp/BDDnaJ‐OE 28 days post‐inoculation (dpi). (b) Local symptoms of internode leaves of ΔBDDnaJ‐OE and ΔBDClp/BDDnaJ‐OE 28 days after inoculation. (c) Disease index from 0 to 28 days after inoculation in ΔBDDnaJ‐OE and ΔBDClp/BDDnaJ‐OE. Significant difference, *p < 0.05. (d–h) Changes in catalase (CAT), superoxide dismutase (SOD), chlorophyll, H₂O₂, and salicylic acid (SA) content before and after inoculation in ΔBDDnaJ‐OE and ΔBDClp/BDDnaJ‐OE. (i) Subcellular localisation of mCherry‐BDDnaJ and GFP‐BDClp under GFP, mCherry, bright field, and merge conditions. Different lowercase letters indicate statistically significant differences after the Waller–Duncan test (α = 0.05). Silenced: ΔBDDnaJ‐RNAi, ΔBDClp‐RNAi; overexpression: ΔBDDnaJ‐OE, ΔBDClp‐OE.

FIGURE 6

BDClp cooperates with BDDnaJ in regulating the unfolded protein response (UPR). (a) Symptoms of wild‐type Bambusa pervariabilis × Dendrocalamopsis grandis (WT), ΔBDDnaJ‐RNAi, and ΔBDClp‐RNAi after dithiotreitol (DTT) treatment. (b) Expression pattern of UPR‐related genes in wild‐type, ΔBDDnaJ‐RNAi, and ΔBDClp‐RNAi after DTT treatment. (c) Differential growth of wild‐type and transgenic Nicotiana benthamiana ΔBDClp‐RNAi and ΔBDDnaJ‐RNAi after root irrigation with DTT. (d) Bar graph showing biomass measurements of wild‐type and transgenic N. benthamiana ΔBDClp‐RNAi and ΔBDDnaJ‐RNAi after root irrigation with DTT for 7 days. (e) Staining of reactive oxygen species (ROS) in wild‐type and transgenic N. benthamiana ΔBDClp‐RNAi and ΔBDDnaJ‐RNAi after DTT treatment. (f) Bar graph showing ROS content measurements of wild‐type and transgenic N. benthamiana ΔBDClp‐RNAi and ΔBDDnaJ‐RNAi after DTT treatment. (g) Transmission electron microscopy images of wild‐type and transgenic N. benthamiana ΔBDClp‐RNAi and ΔBDDnaJ‐RNAi treated with DTT. Different lowercase letters indicate statistically significant differences after the Waller–Duncan test (α = 0.05). Silenced: ΔBDDnaJ‐RNAi, ΔBDClp‐RNAi; overexpression: ΔBDDnaJ‐OE, ΔBDClp‐OE.

FIGURE 7

BDDnaJ regulates BDBiP protein stability by interacting with BDBiP. (a) Prediction of BDDnaJ interaction sites with BDBiP, ZIP60, PDI proteins. (b) Yeast two‐hybrid interaction of BDDnaJ was tested against BDBiP, ZIP60 and PDI. (c) Quantification of NbBiP proteins in dithiothreitol (DTT)‐treated ΔNbDnaJ‐OE and ΔNbDnaJ‐RNAi using Nicotiana benthamiana wild type (WT) as a control in a western blot assay with greyscale values shown. (d) Relative conductivity assay in DTT‐treated ΔBDDnaJ‐OE and ΔBDDnaJ‐RNAi using WT as control. (e) Relative conductivity assay in DTT‐treated ΔBDDnaJ‐OE and ΔBDDnaJ‐RNAi treated with HA15 beforehand and then with WT as control. Different lowercase letters indicate statistically significant differences after the Waller–Duncan test (α = 0.05). Silenced: ΔBDDnaJ‐RNAi, ΔBDClp‐RNAi; overexpression: ΔBDDnaJ‐OE, ΔBDClp‐OE.