Bacterial type III effector-induced plant C8 volatiles elicit antibacterial immunity in heterospecific neighbouring plants via airborne signalling

Abstract

Upon sensing attack by pathogens and insect herbivores, plants release complex mixtures of volatile compounds. Here, we show that the infection of lima bean (Phaseolus lunatus L.) plants with the non-host bacterial pathogen Pseudomonas syringae pv. tomato led to the production of microbe-induced plant volatiles (MIPVs). Surprisingly, the bacterial type III secretion system, which injects effector proteins directly into the plant cytosol to subvert host functions, was found to prime both intra- and inter-specific defense responses in neighbouring wild tobacco (Nicotiana benthamiana) plants. Screening of each of 16 effectors using the Pseudomonas fluorescens effector-to-host analyser revealed that an effector, HopP1, was responsible for immune activation in receiver tobacco plants. Further study demonstrated that 1-octen-3-ol, 3-octanone and 3-octanol are novel MIPVs emitted by the lima bean plant in a HopP1-dependent manner. Exposure to synthetic 1-octen-3-ol activated immunity in tobacco plants against a virulent pathogen Pseudomonas syringae pv. tabaci. Our results show for the first time that a bacterial type III effector can trigger the emission of C8 plant volatiles that mediate defense priming via plant-plant interactions. These results provide novel insights into the role of airborne chemicals in bacterial pathogen-induced inter-specific plant-plant interactions.

Keywords: 1-octen-3-ol; C8 volatiles; airborne defense; lima bean; plant-plant interactions; self- and non-self-recognition; tobacco; type III effector.

Figure 1

Schematic representation of the experimental design. In an acrylic chamber (width, length and height = 55 × 95 × 65 cm³), a 3‐week old lima bean (emitter) plant was inoculated with Pseudomonas syringae pv. tomato DC3000 (Pto) to induce volatile production. Tobacco (receiver) plants placed in the same chamber were exposed to these volatiles for 1 week and then challenged with Pseudomonas syringae pv. tabaci (Pta). The activation of disease resistance in receiver plants by the airborne signalling molecules was evaluated by assessing the Pta population size [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Bacterial type III effector‐induced plant volatile emission in the emitter plant elicits immunity in the neighbouring receiver plants. (a) Pathogen population in receiver plants. Tobacco (receiver) plants were exposed to volatiles released by the Pto‐inoculated lima bean (emitter) plant for 7 days. The Pta population sizes in receiver plants that were pre‐exposed to Pto and non‐exposed control plants were measured at 0 and 3 dpi with Pta. The hrpL mutant was used as a negative control to study the function of the effector. The represent pictures regarding Pta resistance in Pto pre‐exposed tobacco leaf are provided in Figure S4 (b) Expression analysis of disease resistance genes, NbPR1a, NbPR1c, NbHIN1 and NbSAR8.2, in receiver and control plants by quantitative reverse‐transcription PCR (qRT‐PCR) at 0 and 6 hpi. (c) Schematic of the delivery system used to stably introduce individual type III effectors from Pto into plant cells using P. fluorescens (Pf) EtHAn, a non‐pathogenic strain with no native effector. IM: inner membrane; OM: outer membrane; PM: plasma membrane. Data represent mean ± standard error (SE; n = 4 plants per treatment). Different letters indicate significant differences between treatments (p < .05; least significant difference [LSD] test). The experiment was repeated three times [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Bacterial type III effector‐mediated immune activation in receiver plants via emitter plant‐derived volatiles. Pathogen populations were measured in tobacco (receiver) plants at 3 dpi with Pta (OD₆₀₀ = 1). Emitter plants were treated with P. fluorescens (Pf) EtHAn expressing various bacterial effectors to induce effector‐mediated volatile compound emission. Data represent mean ± SE (n = 4 plants per treatment). Different letters indicate significant differences between treatments (p = .05; LSD). E.V.: empty vector. The experiment was repeated three times [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Identification of volatile compounds produced by lima bean plants upon treatment with the bacterial effector HopP1. (a) Gas chromatography (GC) profiles of volatiles emitted from lima bean plants at 72 hpi after treatment with wild‐type Pto, Pf EtHAn HopP1, Pf EtHAn and control. (b–d) Peak areas measured for the three C8 volatiles, 1‐octen‐3‐ol (b), 3‐octanone (c), and 3‐octanol (d), at 72 hpi with Pto, Pto hrcC‐, Pto hrpL‐, Pf EtHAn HopP1, Pf EtHAn and non‐treated control. Data represent mean ± SE (n = 4 plants per treatment). Different letters indicate significant differences between treatments (p = .05; LSD test). The experiment was repeated three times [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Effect of 1‐octen‐3‐ol, a keynote volatile, on the immunity of receiver plants. (a) Representative photograph showing Pta‐induced symptoms on tobacco (receiver) plants at 7 dpi. Prior to pathogen infiltration, plants were exposed to varying concentrations of 1‐octen‐3‐ol for 7 days. (b) Quantification of pathogen population at 0 and 3 dpi with Pta in receiver plants exposed to different concentrations of 1‐octen‐3‐ol. (c) Expression levels of NbPR1a, NbPR1c, NbHIN1 and NbSAR8.2 in tobacco plants exposed to 1‐octen‐3‐ol. Gene expression was analysed by quantitative real‐time polymerase chain reaction (qRT‐PCR) at 0, 6 and 24 hpi. (d) Quantification of pathogen population in SABP2‐ and CO1‐silenced tobacco plants treated with 1‐octen‐3‐ol or water (control). After treatment with 1‐octen‐3‐ol or water for 7 d, Pta (10⁸ cfu/ml) was infiltrated into tobacco leaves, and pathogen population size was measured at 0 and 3 dpi. Data represent mean ± SE (n = 5 plants per treatment). Different letters indicate significant differences between treatments (p = .05; LSD test) in each silencing plant. The experiment was repeated three times [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Proposed mechanism of bacterial type III effector‐dependent airborne immune activation. A lima bean (emitter) plant was challenged with the non‐host pathogen, Pto, which delivered the effector protein HopP1 into the plant cells through the T3SS. The HopP1 protein potentially interacts with plant protein(s) involved in the emission of C8 plant volatiles. Unknown proteins (or enzymes) generate C8 volatiles, including 1‐octen‐3‐ol, which travel to tobacco (receiver) plants through air. The perception of C8 plant volatiles by tobacco plants activates the expression of pathogenesis‐related (PR) genes and subsequently induces systemic acquired resistance (SAR) against Pta through the salicylic acid (SA)‐dependent signalling pathway, thereby suppressing Pta growth. Arrows indicate a multi‐organism interaction cascade that leads to disease resistance in neighbouring plants [Colour figure can be viewed at wileyonlinelibrary.com]