Plants have evolved a powerful immune system to resist their potential colonization by microbial pathogens and parasites. Over the past decade, it has become increasingly clear that this innate immunity is, in essence, composed of two interconnected branches, termed PAMP-triggered immunity and effector-triggered immunity. PTI is triggered by recognition of pathogen- or microbial-associated molecular patterns , which are conserved molecular signatures decorating many classes of microbes, including non-pathogens. Perception of MAMPs by pattern recognition receptorsat the cell surface activates a battery of host defense responses leading to a basal level of resistance. As a result of the evolutionary arms-race between plants and their intruders, many microbial pathogens acquired the ability to dodge PTI-based host surveillance via secretion of effector molecules that intercept MAMP triggered defense signals. In turn, plants have adapted to produce cognate R- proteins by which they recognize, either directly or indirectly, these pathogen specific effector proteins, resulting in a superimposed layer of defense variably termed effector-triggered immunity , gene-for-gene resistance or R-gene-dependent resistance. In many cases, effector recognition culminates in the programmed suicide of a limited number of challenged host cells, clearly delimited from the surrounding healthy tissue. This hypersensitive responseis thought to benefit the plant by restricting pathogen access to water and nutrients and is correlated with an integrated set of physiological and metabolic alterations that are instrumental in impeding further pathogen ingress, among which a burst of oxidative metabolism leading to the massive generation of reactive oxygen species. Apart from local immune responses, ETI-associated HR formation also mounts a long-distance immune response termed systemic acquired resistance , in which naïve tissues become resistant to a broad spectrum of otherwise virulent pathogens.
It should be noted, however, that PTI, when activated by PAMPs that activate the SA signaling pathway,ebb and flow bench can trigger SAR as well. An archetypal inducible plant defense response, SAR requires endogenous accumulation of the signal molecule salicylic acidand is marked by the transcriptional reprogramming of a battery of SA-inducible genes encoding pathogenesis-related proteins. By contrast, there is ample evidence for induced disease resistance conditioned by molecules other than SA, as illustrated by rhizobacteria-mediated induced systemic resistance [ISR; [9]]. ISR, which delivers systemic protection without the customary pathogenesis-related protein induction, is a resistance activated upon root colonization by specific strains of plant growth-promoting rhizobacteria. In a series of seminal studies using the reference strain Pseudomonas fluorescens WCS417r, Pieterse and associates demonstrated that, at least in Arabidopsis, ISR functions independently of SA, but requires components of the jasmonic acidand ethylene response pathways. Even though colonization of the roots by ISR-triggering bacteria leads to a heightened level of resistance against a diverse set of intruders, often no defense mechanisms are activated in above ground plant tissues upon perception of the resistance-inducing signal. Rather, these tissues are sensitized to express basal defense responses faster and/or more strongly in response to pathogen attack, a phenomenon known as priming. As demonstrated recently, priming of the plant’s innate immune system confers broad-spectrum resistance with minimal impact on seed set and plant growth. Hence, priming offers a cost-efficient resistance strategy, enabling the plant to react more effectively to any invader encountered by boosting infection-induced cellular defense responses. In contrast to the overwhelming amount of information on inducible defenses in dicotyledonous plant species, our understanding of the molecular mechanisms underpinning induced disease resistance in rice and other cereals is still in its infancy.
Evidence demonstrating that central components of the induced resistance circuitry, including the master regulatory protein NPR1, are conserved in rice has only recently been presented. Moreover, reports on SAR-like phenomena in rice are scarce. Most tellingly in this regard, a 17- year-old report of systemically enhanced resistance against the rice blast pathogen M. oryzae triggered by a localized infection with the non-rice pathogen P. syringae pv. syringae remains one of the most compelling examples of a SAR-like response in rice to date. In contrast, there is a sizeable body of evidence demonstrating systemic protection against various rice pathogens resulting from ISR elicited by, amongst others, Pseudomonas, Bacillus and Serratia strains. However, in most if not all cases, still very little is known about the basic mechanisms governing this ISR response. In a previous report, we demonstrated that rice plants of which the roots were colonized by the fluorescent pseudomonad P. aeruginosa 7NSK2 developed an enhanced defensive capacity against infection with M. oryzae. Bacterial mutant analysis revealed that this 7NSK2-mediated ISR is based on secretion of the redox-active pigment pyocyanin. Perception of pyocyanin by the plant roots was shown to cue the formation of reiterative micro-oxidative bursts in naïve leaves, thereby priming these leaves for accelerated expression of HR-like cell death upon pathogen attack. Aiming to gain further insight into themolecular mechanisms underpinning rhizobacteria-modulated ISR in rice, we tested the ability of the biocontrol agent Serratia plymuthica IC1270 to induce systemic resistance against various rice pathogens with different modes of infection. Originally isolated from the rhizosphere of grapes,S. plymuthica IC1270 is a well-characterized PGPR strain producing a broad palette of antimicrobial compounds. In addition to its potential as a direct antagonist of a wide array of plant pathogens, preliminary experiments in bean and tomato revealed that IC1270 is equally capable of reducing disease through activation of a plant-mediated defense response. Here, we demonstrate that colonization of rice roots by IC1270 renders foliar tissues more resistant to M. oryzae.
Using a combined cytological and pharmacological approach, evidence is provided that IC1270 locks plants into a pathogen-inducible program of boosted ROS formation, culminating in the prompt execution of HR cell death at sites of attempted pathogen entry. Similar, yet even more pronounced, phenotypes of hypersensitively dying cells in the vicinity of fungal hyphae were observed in a genetically incompatible rice-M. oryzae interaction, suggesting that IC1270-mediated ISR and R-gene-mediated ETI involve similar defense mechanisms. Bacterial strains used in this study were Serratia plymuthica IC1270, which was originally described as Enterobacter agglomerans, and Pseudomonas aeruginosa 7NSK2. For inoculation experiments, IC1270 and 7NSK2 were grown on iron-limiting King’s B medium [KB; [34]] for 24 h at 28°C and 37°C, respectively. Bacterial cells were scraped off the plates and suspended in sterile saline . Densities of the bacterial suspensions were adjusted to the desired concentration based on their optical density at 620 nm. Magnaporthe oryzae isolate VT7, a field isolate from rice in Vietnam, was grown at 28°C on half-strength oatmeal agar . Seven-day-old mycelium was flattened onto the medium using a sterile spoon and exposed to blue light for seven days to induce sporulation. Conidia were harvested as described in De Vleesschauwer et al., and inoculum concentration was adjusted to a final density of 1 × 104 spores ml-1 in 0.5% gelatin .Rhizoctonia solani isolate MAN-86, belonging to anastomosis group AG-1 IA, was maintained on potato dextrose agar . Inoculum was obtained according to Rodrigues et al. with minor modifications. After autoclaving, 15 toothpicks, 1 cm in length, and five agar plugs , obtained from the margin of an actively growing colony of R. solani, were transferred to PDA plates. These plates were then incubated for 8 days at 28°C so R. solani could colonize the toothpicks. Cochliobolus miyabeanus strain 988, obtained from diseased rice in field plots at the International Rice Research Institute , was grown for sporulation at 28°C on PDA. Seven-day-old mycelium was flattened onto the medium using a sterile spoon and exposed to blue light for three days under the same conditions mentioned above. Upon sporulation, conidia were harvested exactly as stated in Thuan et al. and re-suspended in 0.5% gelatin to a final density of 1 × 104 conidia ml-1.Four-week-old rice seedlings were challenge inoculated with Magnaporthe oryzae as described in De Vleesschauwer et al.. Six days after inoculation, disease severity on the fourth leaves of each plant was rated by counting the number of elliptical to round-shaped lesions with a sporulating gray center, and expressed relative to non-induced control plants. R. solani bioassays were performed essentially as described in Rodrigues et al.. Plants were challenged when four weeks old by placing a 1-cm toothpick colonized by R. solani inside the sheath of the second youngest fully expanded leaf. Inoculated plants were maintained inside humid inoculation chambersfor 72 h, and, thereafter,4x8ft rolling benches transferred to greenhouse conditions. Four days after challenge infection, disease severity was assessed by measuring the length of the water-soaked lesions. C. miyabeanus bio-assays were performed as described in Ahn et al. with minor modifications. Five-week-old seedlings were misted with a C. miyabeanus spore suspension containing 1 × 104 conidia ml-1 in 0.5% gelatin. Inoculated plants were kept in a dew chamber for 18 h to facilitate fungal penetration, and subsequently transferred to greenhouse conditions for disease development. Disease symptoms were scored at four days after inoculation for about 48 leaves per treatment. Disease ratings were expressed on the basis of diseased leaf area and lesion type: I, no infection or less than 2% of leaf area infected with small brown specs less than 1 mm in diameter; II,less than 10% of leaf area infected with brown spot lesions with gray to white center, about 1–3 mm in diameter; III, average of about 25% of leaf area infected with brown spot lesions with gray to white center, about 1–3 mm in diameter; IV, average of about 50% of leaf area infected with typical spindle-shaped lesions, 3 mm or longer with necrotic gray center and water-soaked or reddish brown margins, little or no coalescence of lesions; V, more than 75% of leaf area infected with coalescing spindle-shaped lesions.Induced systemic resistance assays were performed as described in De Vleesschauwer et al. with minor modifications.
Briefly, rice plants were grown under greenhouse conditions in commercial potting soil that had been autoclaved twice on alternate days for 21 min. Rice seeds first were surface sterilized with 1% sodium hypochlorite for two min, rinsed three times with sterile, demineralized water and incubated for five days on a wet sterile filter paper in sealed Petri dishes at 28°C. Prior to sowing in perforated plastic trays , roots of germinated seeds were dipped in a bacterial suspension of the ISR-inducing strains [5 × 107 colony-forming unitsml-1] for 10 min. The auto claved soil was thoroughly mixed with bacterial inoculum to a final density of 5 × 107 cfu ml-1. To ensure consistent root colonization by the eliciting bacteria, rice plants were soil-drenched a second time with bacterial inoculumat ten days after sowing. In control treatments, soil and rice plants were treated with equal volumes of sterilized saline. For experiments in which purified pyocyanin was applied to the roots of rice seedlings, plants were grown in a hydroponic gnotobiotic system as described before. In this system, plants were fed with various concentrations of pyocyanin and ascorbate 4 days before challenge inoculation by adding the desired concentration to the half-strength Hoagland nutrient solution. Pyocyanin extraction, quantification and application were performed exactly as stated in De Vleesschauwer et al..To gain more insight into the nature of IC1270-mediated ISR against M. oryzae, cytological studies were performed at sites of pathogen entry. To this purpose, we adopted the intact leaf sheath assay previously described by Koga et al.. Briefly, leaf sheaths of the fifth leaf of rice plants at the 5.5 leaf stage were peeled off with leaf blades and roots. The leaf sheath was laid horizontally on a support in plastic trays containing wet filter paper, and the hollow space enclosed by the sides of the leaf sheaths above the mid vein was filled with a suspension of sporesof M. oryzae. Inoculated leaf sheaths were then incubated at 25°C with a 16-h photoperiod. When ready for microscopy, the sheaths were hand-trimmed to remove the sides and expose the epidermal layer above the mid vein. At least five trimmed sheath tissue sections originating from different control and IC1270-treated plants were used for each sampling point. Phenolic compounds were visualized as autofluorescence under blue light epifluorescence . To detect H2O2 accumulation, staining was performed according to the protocol of Thordal-Christensen et al. with minor modifications. Six hours before each time point, trimmed sheath segments were vacuum-infiltrated with an aqueous solution of 1 mg ml-1 3,3′-diaminobenzidine-HCLfor 30 min. Thereafter, infiltrated segments were incubated in fresh DAB solution until sampling.