DAB polymerizes in the presence of H2O2 and endogenous peroxidase to form a brownish-red precipitate that can be easily visualized using bright-field microscopy. After staining, trimmed sheath segments were mounted in 50% glycerol. Images were acquired digitally and further processed with the Olympus analySIS cell^F software.To assess the ISR-triggering capacity of S. plymuthica IC1270, susceptible rice plants were grown in soil containing IC1270 bacteria, and subsequently challenged with several fungal pathogens exhibiting different modes of infection. In these ISR bio-assays, the resistance-inducing potential of IC1270 was compared to that of P. aeruginosa 7NSK2, a well-studied PGPR strain which we previously uncovered as a potent activator of induced resistance responses in rice. We first tested whether root colonization by S. plymuthica IC1270 exerts a protective effect against infection by the hemibiotrophic ascomycete M. oryzae, causal agent of the devastating rice blast disease and a major threat to food security worldwide. By 4 days post-inoculation , leaves of control, non-induced plants displayed typical water-soaked, diamond-shaped lesions, developing conidia at the center of each lesion by 6 dpi. In contrast, IC1270-colonized plants exhibited a marked reduction in the number of these susceptible-type lesions, producing a resistance phenotype mimicking that of quantitative trait loci-governed intermediate resistance . This resistance type is characterized by the abundance of small necrotic non-sporulating lesions,flood and drain table less than 2 mm in diameter, 60 to 72 h post-inoculation . Consistent with our previous findings, treatment with P. aeruginosa 7NSK2 resulted in a substantial reduction of disease as well.
No significant differences in the number of susceptible-type lesions could be observed between IC1270- and 7NSK2-treated plants, indicating that IC1270 and 7NSK2 are equally effective in suppressing M. oryzae. Because IC1270 clearly inhibited the growth of M. oryzae in dual culture experiments , possible systemic plant colonization by the rhizobacteria was checked. However, in all bio-assays performed, IC1270 bacteria were absent from sheaths or leaves of root induced plants, indicating that bacterial colonization remained confined to the root zone . Although such spatial separation does not rule out the possibility that IC1270-conferred protection might result from long-distance translocation of bacteria-produced allelochemicals to systemic leaves, the latter is rather unlikely as pilot experiments aimed at elucidating the bacterial traits underpinning IC1270-ISR revealed that mutants defective in the global response regulator protein GacA, which controls the synthesis of various anti-fungal metabolites , were as effective as wild-type IC1270 in reducing rice blast disease severity . The cumulative data therefore strongly suggest that the beneficial protective activity exerted by S. plymuthica IC1270 is based on activation of the plant’s defensive repertoire, rather then being caused by microbial antagonism. To test the spectrum of effectiveness of this IC1270-mediated ISR, we next assayed for induction of resistance against the sheath blight pathogen, Rhizoctonia solani, and the brown spot pathogen, Cochliobolus miyabeanus, both of which are considered necrotrophic fungi. In contrast to M. oryzae, which sequentially invades living cells, R. solani and B. oryzae kill host cells at very early stages in the infection, leading to extensive tissue damage. As shown in Fig 1B, both IC1270 and 7NSK2 failed to reduce disease caused by R. solani.
This impaired ISR response was not due to insufficient root colonization as bacterial counts in the rhizosphere of treated rice seedlings were comparable to those obtained in the M. oryzae bio-assays . Interestingly, in all four independent experiments, IC1270 pretreatment favored subsequent infection by R. solani, causing an average 39.6% increase in disease severity relative to non-induced controls. A similar trend was observed when challenging with C. miyabeanus, with IC1270 consistently promoting vulnerability to the latter pathogen . Root colonization by 7NSK2, however, yielded variable results. No significant differences between control and 7NSK2- treated plants could be observed in three bio-assays, whereas in the two remaining assays, root treatment with7NSK2 rendered rice seedlings substantially more susceptible to brown spot. In all experiments, mock-inoculated control plants remained healthy, and no apparent differences in appearance, size, or weight of control, 7NSK2 or IC1270-treated plants were observed prior to challenge infection . Thus, under the experimental conditions used in this study, root treatment with the ISR-inducing bacteria did not lead to detectable effects on plant growth that could have affected the growth or development of the respective pathogens. Collectively, these findings demonstrate that S. plymuthica IC1270 plays an ambivalent role in the rice induced resistance network, acting as a potent elicitor of resistance to the hemibiotroph M. oryzae while promoting susceptibility to the necrotrophs C. miyabeanus and R. solani.To begin to unravel the defense mechanism underpinning IC1270-mediated ISR, we analyzed the cytological alterations associated with restriction of M. oryzae in IC1270-induced plants using the intact leaf sheath method designed by Koga and associates. In this system, intact leaf sheaths of control, non-induced and IC1270-treated plants of the highly susceptible rice variety CO39 were routinely inoculated by injecting a conidial suspension of the virulent blast isolate VT7.
For comparison with R gene-mediated ETI, we also included the VT7- resistant variety C101LAC, the latter being a near-isogenic line of CO39 carrying the blast resistance genes Pi-1 and Pi-33.Similarly, quantitative recording of attempted blast infections revealed no significant differences in the number of unsuccessful penetration events, indicating that both IC1270-mediated ISR and R-gene-conditioned ETI are unlikely to impede pre-penetration development by M. oryzae . On the other hand, epidermal cells were found to respond to fungal ingress through various cellular reaction types depicted at 48 hpi in Fig 2A. A susceptible reaction was manifested as a type 1 phenotype in which extensively branched invasive hyphae vigorously invaded living epidermal cells with little or no visible host response. Interaction phenotype 2, on the other hand, was characterized by prompt arrest of fungal growth in the first-invaded epidermal cell, a phenomenon associated with enhanced vesicular activity and browning of the anti-clinal cell walls, while a type 3 reaction represented infection sites in which fungal invasion was curtailed shortly after penetration due to development of HR-like cell death, as indicated by the characteristic aggregation of the cytoplasm and a bright autofluorescence of the anticlinal cell walls. As expected, sheath cells of noninduced, susceptible CO39 plants inoculated with virulent VT7 predominantly mounted a type 1 reaction, whereas HR was the prevailing plant response in the incompatible interaction between VT7 and C101LAC. Most conspicuously, IC1270-induced CO39 sheath cells displayed an interaction profile resembling that observed in VT7-invaded sheaths of genetically resistant C101LAC, with type 3 reactions accounting for approximately 60% of all interactions by 48 hpi . At later stages of infection, M. oryzae had massively colonized the epidermis and mesophyll of CO39 sheaths causing extensive host damage as evidenced by the ubiquitous presence of cellular debris and fragmented remnants of host cell walls around invasive hyphae in the mesophyll . By contrast, in resistant C101LAC, as well as in IC1270-induced CO39, invading hyphae were largely trapped within hypersensitively dying cells in the epidermal layer, preventing fungal passage to the underlying tissue. Because rapid accumulation of phenolic compounds is a hallmark of rice defense against M. oryzae, we also examined the effect of IC1270 pre-treatment on the level of autofluorescence. Autofluorescence was detectable as early as 18 hpi, irrespective of IC1270 treatment or the level of resistance of the cultivars used . However, similar to what was observed in resistant C101LAC,rolling bench root treatment of CO39 with IC1270 caused the frequency of autofluorescent appressorial sites to increase rapidly from 18 hpi onward, reaching a level of 60 and 100% of all interactions by 24 and 36 hpi, respectively . By contrast, in non-induced CO39 cells, less than 6% of the appressorial sites showed autofluorescence 24 hpi, indicating that root colonization by IC1270 primes rice sheath cells for accelerated deposition of autofluorescent phenolic compounds at sites of attempted pathogen invasion. Along with the high frequency of hypersensitively reacting cells, these observations suggest that IC1270- mediated ISR and R-gene-conditioned ETI act, at least in part, through a similar set of defense reactions.There is ample evidence demonstrating the active involvement of reactive oxygen species , and H2O2 in particular, in the induction, signaling and execution of blast resistance in rice.
Furthermore, in the course of previous studies, we demonstrated that pyocyanininduced H2O2 micro-bursts are primordial for the onset of P. aeruginosa 7NSK2-mediated ISR against M. oryzae. Taking these facts into account, we sought to extend ourcytological analysis of ISR elicited by IC1270 by monitoring the spatiotemporal patterns of pathogenesis-related H2O2 production. In planta accumulation of H2O2 was visualized using an endogenous peroxidase-dependent staining procedure with 3,3′-diaminobenzidine . In these DAB assays, reddish-brown precipitates are deposited at the sites of H2O2 accumulation. No DAB accumulation was observed in mock-inoculated controls, regardless of IC1270 treatment or the inherent level of resistance of the cultivars used. However, comparative analysis of H2O2 production in pathogen-inoculated seedlings revealed the occurrence of a wide range of distinct DAB staining patterns that could be grouped into five categories . The first type comprised interaction sites in which DAB accumulation was not detectable despite massive fungal colonization of both penetrated and neighboring epidermal cells. Conversely, interaction sites displaying H2O2 accumulation in the primary invaded epidermal cell following spread of the invasive hyphae into neighboring cells were classified as a type II reaction. Type III interaction sites were characterized by the ubiquitous occurrence of DAB-positive vesicle-like bodies targeted to the invading hyphae. A type IV reaction referred to intracellular DAB staining tightly associated with the characteristic cytoplasmic aggregates of HR-expressing cells , while interaction sites displaying wholecell DAB accumulation were scored as a type V reaction. Importantly, when the DAB solution was supplemented with ascorbate, staining was abolished, indicating that the staining was due to H2O2 . Leaf sheath cells of susceptible CO39 were characterized by the high ratio of H2O2-negative type I reactions, accounting for 78% and 67% of all interaction sites by 36 and 48 hpi, respectively . In some incidences , H2O2accumulated in the initially penetrated epidermal cell following the formation of an extensively branched mycelium in the neighboring cells. Yet, this type II reaction seemingly occurred too late to effectively stall the pathogen. IC1270-induced CO39 cells, on the other hand, exhibited a strikingly different set of responses in that type I reactions, reaching a level of 33% at 36 hpi, were no longer discernible by 48 hpi. The rapid decline in the frequency of type I reactions from 36 hpi onward corresponded to an approximately 15% increase in the frequency of both type III and type V reactions. HR-like cell death of attacked epidermal cells, seen at approximately 52% of all interaction sites, was always associated with H2O2 accumulation in the cytoplasmic aggregates, beginning 32 hpi. Although not identical, by 48 hpi the H2O2 signature of IC1270-treated CO39 plants showed substantial similarity to that observed in the incompatible interaction between C101LAC and VT7, thereby further emphasizing the possible mechanistic similarities between IC1270-mediated ISR and R-protein-dictated ETI. Starting 50 hpi, a strong accumulation of H2O2 was found in CO39 mesophyll cells that appeared to collapse, whereas in samples from IC1270-induced CO39 or C101LAC sheaths, DAB staining in the mesophyll layer was seldom observed . However, at these late infection stages, massive H2O2 accumulation is most likely a consequence of progressive cellular destruction and overtaxed anti-oxidative capacities, and hence, a chaotic reaction associated with susceptibility, rather than a controlled defense response restricting cellular accessibility for M. oryzae. Together these results clearly demonstrate the potential of IC1270 to prime rice for augmented generation of epidermis-localized H2O2.In light of the well-documented ability of ROS to serve multiple defense-related signaling functions, sometimes with opposite effects in different contexts, we asked whether the ability of IC1270 to boost pathogenesis-related H2O2 generation might account for the differential effectiveness of IC1270-mediated ISR against M.oryzae, R. solani and C. miyabeanus. To address this question, we examined the effect of manipulating the oxidative stress in pathogen-inoculated leaves on subsequent disease development. To artificially raise the level of ROS in inoculated leaves, detached leaves were pressure-infiltrated with mixtures of glucose plus glucose oxidaseand xanthine plus xanthine oxidase .