Magnaporthe oryzae, an ascomycete fungus, is the causal agent of rice blast disease and is responsible for 10-40% of global rice yield losses, causing an estimated $66 billion in economic damage annually (Nalley et al., 2016; Talbot, 2003; Wilson and Talbot, 2009). The disease threatens food security, as the rice lost to blast could feed up to 60 million people each year (Nalley et al., 2016). M. oryzae initiates infection by forming a specialized structure called the appressorium, which generates enormous turgor pressure to breach the plant cuticle (Cruz-Mireles et al., 2021; de Jong et al., 1997; Eseola et al., 2021; Howard and Valent, 1996). After penetration via a narrow penetration peg, the fungus develops invasive hyphae (IH) and establishes a biotrophic interfacial complex, allowing the secretion of effector proteins that suppress plant defenses and facilitate colonization (Cruz-Mireles et al., 2021; Oliveira-Garcia et al., 2023, 2024). Among the secreted effectors, the MAX (Magnaporthe Avrs and ToxB-like) family has been characterized by a conserved β-sandwich structure stabilized by disulfide bonds, despite high sequence divergence (de Guillen et al., 2015; Lahfa et al., 2024). MAX effectors are recognized by nucleotide-binding leucine-rich repeat (NLR) immune receptors via integrated heavy metal-associated (HMA) domains, triggering effector-triggered immunity (ETI) and hypersensitive response (HR) cell death (Guo et al., 2018; Liu et al., 2021; Varden et al., 2019). The evolutionary conservation of MAX effector structures suggests maintenance of critical host-binding functions while allowing sequence diversification to evade recognition. Beyond MAX effectors, recent computational analyses have predicted 195 candidate M. oryzae effectors (MOEs) from diverse M. oryzae lineages based on sequence and structural similarity to known effectors, broadening the repertoire available for functional studies (Petit-Houdenot et al., 2020).
Efforts to develop blast-resistant rice cultivars have focused on introducing resistance (R) genes, such as RGA5 and Pikp-1, which recognize MAX effectors through HMA-mediated interactions (Cesari et al., 2013; Guo et al., 2018; Maqbool et al., 2015). Advances in NLR engineering have expanded effector recognition, but the rapid evolution of pathogen populations necessitates the exploration of more durable strategies, including nonhost resistance (NHR). NHR is a robust, broad-spectrum immune response that protects plants from non-adapted pathogens. It involves multiple layers of defense, including preformed barriers (cuticles, cell walls) and inducible responses such as pattern-triggered immunity (PTI), chemical defenses, and ETI (Oh and Choi, 2022; Wu et al., 2023). PTI is activated through the recognition of pathogen-associated molecular patterns by pattern recognition receptors (PRRs), with BAK1 functioning as a key coreceptor (Chinchilla et al., 2007; Dodds and Rathjen, 2010; Liao et al., 2016; Park et al., 2011). When adapted pathogens deploy effectors to overcome PTI, recognition by intracellular NLRs activates ETI, often culminating in HR (Yuan et al., 2021). Key immune components regulate these processes: SGT1, together with RAR1 and HSP90, stabilizes NLRs and is essential for ETI (Austin et al., 2002; Azevedo et al., 2002; Shirasu, 2009), while salicylic acid (SA) orchestrates both local and systemic defense signaling (Delaney et al., 1994; Thomma et al., 2001). Plants deficient in SA accumulation, such as NahG transgenics, exhibit impaired immune responses and enhanced pathogen susceptibility (Nakao et al., 2011).
To comprehensively investigate the immune responses of N. benthamiana against M. oryzae, we selected representative marker genes corresponding to different defense layers. NbPR1 was chosen as a canonical marker for SA-dependent ETI, NbWRKY22 for PTI/ETI signaling, NbBAK1 for PRR complex formation in PTI, NbCYP71D20 for chemical defense via phytoalexin biosynthesis, NbEAS for capsidiol production, and NbSGT1 for NLR stability and ETI activation. These markers enabled monitoring of immune activation at both PTI and ETI levels during the nonhost interaction. Notably, although M. oryzae devastates monocot hosts like rice, it is unable to infect Solanaceae species such as N. benthamiana. This resistance is thought to result from the long evolutionary divergence (~140-150 million years ago) between monocots and dicots (Chaw et al., 2004), leading to distinct immune system architectures.
In this study, we examined the infection process of M. oryzae on N. benthamiana, monitored the expression of defense-related genes during infection, and functionally dissected the roles of key immune regulators (EAS, BAK1, SGT1, and SA) using virus-induced gene silencing (VIGS) and NahG plants. Furthermore, we performed a large-scale effector screen with 179 MOEs to determine their ability to induce HR-like cell death and evaluated the role of SGT1-dependent ETI in this recognition. Our findings reveal that SGT1- and SA-mediated ETI responses are crucial components of NHR against M. oryzae in N. benthamiana.
Materials and Methods
Plant materials and growth conditions
Rice plants (Oryza sativa cultivars Dongjinbyeo [DJ] and Hwayoungbyeo [HY]) and Nicotiana benthamiana (wild type [WT] and NahG) plants were cultivated in a controlled environment room at 25°C, 70% relative humidity, and a 16-h photoperiod. Magnaporthe oryzae WT strain PO6-6 was cultured on V8 agar medium at 23°C to support both vegetative growth and conidiation. The GFP-tagged derivative PO6-6:GFP was initially cultured on potato dextrose agar (BD Difco, Franklin Lakes, NJ, USA) for vegetative growth at 23°C and subsequently transferred to V8 agar medium to induce conidiation. Cultures for conidiation were incubated for 2 weeks under continuous fluorescent light. Aerial mycelia were removed by scraping with a spreader 4 days prior to conidia harvesting.
Inoculation and disease evaluation of M. oryzae in plant hosts
Conidia of WT PO6-6 and PO6-6:GFP strains were harvested from V8 agar cultures using distilled water containing 0.025% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). The conidial suspension was adjusted to a final concentration of 2 × 104 conidia/mL using a hemocytometer. For inoculation, 10 μL of the freshly prepared suspension was applied to detached N. benthamiana leaves (the 3rd to 4th leaves from the top of 4- to 5-week-old plants) or rice leaf sheaths. For rice infection assays, M. oryzae PO6-6 conidia (2 × 104 conidia/mL) were drop-inoculated onto detached leaves of O. sativa cultivars DJ and HY. Inoculated samples were incubated in a dew chamber at 24°C in the dark with 100% relative humidity: 48 h for rice and 8 days for N. benthamiana. HR cell death in rice was assessed at 2 days post-inoculation (dpi) using a rice leaf sheath assay, ethanol clearing was performed after infection to enhance visualization of HR-associated lesions. Disease severity was evaluated using a four-grade scoring system for N. benthamiana and by categorizing rice infections into viable IH or HR cell death. Chlorophyll was removed by incubating samples in 100% ethanol to enhance visualization. All experiments were independently repeated at least three times.
Constructs for VIGS and effector screening
For VIGS, 300-bp fragments specific to the target genes (NbEAS, NbBAK1, and NbSGT1) were amplified by PCR. Single gene silencing was achieved using individual fragments, whereas double and triple gene silencing constructs were generated by tandem amplification. All fragments were subsequently cloned into the pTRV2 vector, following the protocol described by Kim et al. (2017). For high-throughput effector screening, 179 candidate MOEs, selected based on successful reconstitution among 195 candidates identified via in silico secretome analyses (Petit-Houdenot et al., 2020), were cloned into a PVX-based expression vector (pKW) using a ligase-independent cloning strategy (Oh et al., 2010). All recombinant constructs were introduced into Agrobacterium tumefaciens GV3101 for subsequent agroinfiltration assays in N. benthamiana.
VIGS in N. benthamiana
Agrobacterium tumefaciens GV3101 strain carrying the following constructs—pTRV1, pTRV2:PDS, pTRV2:GFP, pTRV2:EAS, pTRV2:BAK1, pTRV2:SGT1, pTRV2:EAS:BAK1, pTRV2:EAS:SGT1, pTRV2:BAK1:SGT1, and pTRV2:EAS:BAK1:SGT1—were prepared for agroinfiltration. For the empty vector (EV) control, the pTRV2:GFP construct was used, which contains a nonfunctional fragment of GFP and results in minimal expression of an irrelevant protein without biological significance. Bacterial cells were resuspended in infiltration buffer (10 mM MES, 10 mM MgCl2, and 150 μM acetosyringone, pH 5.6). Suspensions of pTRV1 and the respective pTRV2 constructs were mixed at a 1:1 ratio, and the final OD600 was adjusted to 0.2 for each construct. This mixture was infiltrated into 2-week-old N. benthamiana plants. Three weeks post-infiltration, the sixth leaf was harvested for subsequent experiments. The relative expression levels of silenced genes were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using primers listed in Supplementary Table 1.
Microscopic imaging
Fluorescence microscopy was performed with a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). IH expressing GFP from the PO6-6:GFP strain were visualized using an excitation filter (450-490 nm) and an emission filter (515-565 nm).
Gene expression assay
N. benthamiana leaves were inoculated with M. oryzae PO6-6. Leaf discs (7 mm diameter) were collected daily from 0 to 10 dpi at the inoculation sites and immediately flash-frozen in liquid nitrogen. Total RNA was extracted using TRIzol reagent (MRC, Cincinnati, OH, USA), and 1 μg of RNA was reverse-transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). For qRT-PCR, 50 ng of cDNA was amplified with gene-specific primers (Supplementary Table 1) using Power SYBR Green supermix (Applied Biosystems, Waltham, MA, USA) on a Bio-Rad CFX96 real-time PCR system. Relative transcript levels were normalized to N. benthamiana elongation factor 1-alpha (NbEF1α) as an internal control gene.
Results
M. oryzae invasion induces HR cell death in nonhost N. benthamiana
To examine the host responses to M. oryzae infection, disease progression was monitored in the susceptible rice cultivar DJ and the resistant cultivar HY following inoculation with strain PO6-6. In DJ, penetration pegs and primary IH developed from appressoria, and secondary IH expanded into adjacent cells. In HY, HR cell death was observed at 2 dpi, restricting fungal spread (Fig. 1A). N. benthamiana is considered a nonhost for M. oryzae. To assess the infection process in N. benthamiana, leaves were inoculated with PO6-6 and monitored at 2-day intervals for 10 days. Appressorium formation was observed at 2 dpi, followed by the development of primary IH at 4 dpi. By 6 dpi, localized brown discoloration appeared near the appressorium, indicative of confined cell death. This brownish region expanded within the infected cell by 8 dpi and remained localized by 10 dpi, coinciding with IH growth (Fig. 1B). To characterize the immune responses triggered by M. oryzae in N. benthamiana, we monitored the expression profiles of key defense-related genes over a 10-day infection period. Among the genes examined, NbBAK1, NbEAS, NbWRKY22, and NbPR1 displayed a sustained increase in transcript levels. NbBAK1 showed a gradual increase starting from 1 dpi, while NbEAS exhibited a delayed but steady induction from 3 dpi. NbWRKY22 expression remained low until 8 dpi and then sharply increased, and NbPR1 gradually increased from 1 dpi, peaking at 9 dpi before declining (Fig. 1C). In contrast, NbCYP71D20 and NbSGT1 exhibited dynamic expression patterns, with NbCYP71D20 displaying alternating increases and decreases between 5 and 8 dpi before declining after 9 dpi, and NbSGT1 showing an early induction up to 2 dpi followed by minor fluctuations thereafter (Fig. 1C). These results suggest that both sustained and dynamic activation of immune-related genes contribute to the complex defense response against M. oryzae in N. benthamiana. Based on these differential expression profiles, we next sought to investigate the functional roles of key immune regulators mediating NHR, focusing specifically on NbEAS, NbBAK1, and NbSGT1 due to their notable transcriptional responses and established roles in plant immunity.
SGT1 plays a key role in NHR of N. benthamiana gainst M. oryzae
To assess the roles of NbEAS, NbBAK1, and NbSGT1 in defense against M. oryzae, conidia of PO6-6 were inoculated onto N. benthamiana plants silenced for each target gene, and IH development along with localized cell death were monitored at 8 dpi (Fig. 2A and B), and gene silencing efficiency was validated by qPCR (Fig. 2C). In EV-silenced plants, no IH development was detected, and only a weak brown discoloration was observed after ethanol clearing, similar to WT plants (Fig. 2A). This brown discoloration likely reflects oxidative polymerization of phenolic compounds associated with localized cell death. In plants silenced for NbEAS or NbBAK1 individually, occasional penetration peg formation and limited IH expansion were observed. However, fungal growth remained largely restricted, and HR-like cell death occurred similarly to EV controls (Fig. 2A). In contrast, silencing of NbSGT1, either alone or in combination with NbEAS and/or NbBAK1, led to a markedly different phenotype. Bulbous IH structures consistently developed within infected cells across all NbSGT1-silenced backgrounds (single, double, and triple silencing), indicating enhanced fungal colonization (Fig. 2A). In some cases, expanded IH filled the initially infected cells, although clear movement into adjacent cells was not yet evident at this stage. These findings suggest that NbSGT1 plays a central role in restricting M. oryzae invasion during NHR.
To evaluate the contribution of SA to NHR, similar gene silencing experiments were conducted in NahG plants, which are deficient in SA accumulation. In EV-silenced NahG plants, occasional primary IH development was observed, indicating that reduced SA levels partially compromised basal resistance (Fig. 2B). Strikingly, in NahG plants subjected to NbEAS, NbBAK1, and NbSGT1 triple silencing, fungal invasion was markedly enhanced. Expanded IH structures filled infected cells more extensively, and hyphal growth appeared more aggressive compared to WT plants under the same silencing conditions (Fig. 2B). However, hyphal movement across adjacent cells remained limited under light microscopy at this stage. These observations suggest that both SA-mediated defenses and SGT1-dependent immunity synergistically contribute to restricting initial M. oryzae colonization during nonhost interactions in N. benthamiana.
To visualize hyphal development and assess fungal progression beyond initial infection sites, a GFP-expressing M. oryzae strain (PO6-6:GFP) was utilized under the same gene-silencing conditions (Fig. 3). In EV-silenced WT plants, fluorescence microscopy revealed only appressoria without subsequent IH development, indicating effective restriction of fungal entry. In EV-silenced NahG plants, occasional, primary IH structures were observed within infected cells, suggesting partial compromise of basal defense (Fig. 3A). In contrast, in NbEAS-, NbBAK1-, and NbSGT1-silenced plants, PO6-6:GFP hyphae robustly developed within epidermal cells in both WT and NahG backgrounds. Notably, in NbSGT1 plants subjected to triple gene silencing, GFP-labeled IH extended into adjacent cells, a phenomenon rarely observed in WT plants under similar conditions (Fig. 3B). These results demonstrate combined suppression of SA signaling and SGT1-mediated immunity not only facilitates fungal colonization but also promotes hyphal movement across adjacent cells during nonhost interactions.
Quantification of infection severity and the role of SA in conidiation
To systematically quantify infection severity and assess the effect on fungal reproduction, infection sites were classified into four stages based on the extent of IH development (Fig. 4A). Stage 1 was defined as appressorium formation without visible IH. Stage 2 indicated the presence of bulbous IH confined to the initially infected cell. Stage 3 involved expanded IH filling the invaded cell, And Stage 4 represented hyphal spread into adjacent cells. Quantitative analysis revealed that in WT plants silenced for NbEAS or NbBAK1, infections predominantly remained at Stage 1 or Stage 2, consistent with effective restriction of fungal proliferation (Fig. 4B). In contrast, NbSGT1-silenced WT plants showed a higher proportion of Stage 2 and Stage 3 infections, reflecting enhanced IH development within cells. In NahG plants, occasional progression to Stage 2 was observed in EV-silenced controls, while a substantial proportion of infections progressed to Stage 4 in NahG plants subjected to NbEAS, NbBAK1, and NbSGT1 triple silencing, indicating successful hyphal spread into adjacent cells (Fig. 4B). Furthermore, fungal conidiation was assessed at 8 dpi. While no conidiation was observed in WT plants across all silencing conditions, conidiophores and conidia production were detected in NahG plants subjected to triple silencing (Fig. 4C). These findings suggest that the collapse of both SA-mediated defenses and SGT1-dependent immunity not only facilitates fungal invasion but also supports the completion of the fungal life cycle during nonhost interactions.
SGT1-dependent ETI in N. benthamiana
Having demonstrated that SGT1 and SA signaling are crucial for restricting M. oryzae invasion and proliferation, we next sought to investigate whether SGT1 also plays a role in recognizing pathogen effectors and activating ETI. To this end, a set of 179 candidate MOEs were screened for their ability to induce HR-like cell death in N. benthamiana. Each MOE was cloned into a PVX-based expression vector and delivered into N. benthamiana leaves via Agrobacterium-mediated infiltration along with the P19 silencing suppressor. HR-like cell death symptoms were monitored at 2 dpi. Among the 179 MOEs tested, 70 triggered visible cell death, characterized by localized necrotic lesions at the infiltration sites (Fig. 5A, Supplementary Table 2). This finding indicates that N. benthamiana possesses a diverse set of immune receptors capable of recognizing a wide range of MOEs. To determine whether MOE-induced cell death was dependent on SGT1-mediated immune signaling, eight representative MOEs that induced strong HR cell death were selected for further analysis. When these MOEs were expressed in NbSGT1-silenced plants, cell death responses were significantly compromised compared to EV-silenced controls (Fig. 5B and C). These results demonstrate that SGT1 is essential for effector-triggered cell death activation in N. benthamiana. Collectively, these findings suggest that NHR in N. benthamiana against M. oryzae involves robust recognition of multiple pathogen effectors through SGT1-dependent immune pathways, providing an additional layer of defense beyond basal immunity.
Discussion
Immune gene expression dynamics during M. oryzae infection in N. benthamiana
To investigate the early immune responses activated by Magnaporthe oryzae in Nicotiana benthamiana, we analyzed the temporal expression profiles of key defense-related genes over a 10-day infection period. Distinct transcriptional patterns emerged, reflecting different layers of the immune response. Among the genes examined, NbBAK1, NbEAS, NbWRKY22, and NbPR1 exhibited sustained upregulation, whereas NbCYP71D20 and NbSGT1 showed more dynamic regulation. NbBAK1 gradually increased from 1 dpi, NbEAS was steadily induced from 3 dpi, NbWRKY22 sharply increased after 8 dpi, and NbPR1 showed gradual induction peaking at 9 dpi (Fig. 1A and B). In contrast, NbCYP71D20 fluctuated between 5 and 8 dpi before decreasing after 9 dpi, and NbSGT1 exhibited early transient induction up to 2 dpi followed by stabilization. These expression profiles suggest a complex, multi-layered immune response against M. oryzae in N. benthamiana. Sustained activation of NbBAK1, NbEAS, NbWRKY22, and NbPR1 likely reflects the continuous engagement of basal defense pathways and SA-mediated ETI. In contrast, the dynamic regulation of NbCYP71D20 may reflect finely tuned control of chemical defenses, balancing the production of antimicrobial compounds with host fitness. The transient activation of NbSGT1 likely represents early establishment of NLR-mediated signaling, followed by immune readiness maintenance. Collectively, these findings highlight the temporally regulated and hierarchical nature of immune activation during NHR against M. oryzae.
SGT1 and SA as key mediators of NHR
Functional analysis using VIGS further demonstrated that NbSGT1 and SA are indispensable components of NHR against M. oryzae in N. benthamiana. Silencing of NbEAS or NbBAK1 individually resulted in only limited fungal growth and preserved HR-like cell death responses, whereas silencing of NbSGT1, either alone or in combination with NbEAS and/or NbBAK1, markedly enhanced fungal invasive hyphal development. Similarly, in NahG plants compromised for SA accumulation, triple silencing of NbEAS, NbBAK1, and NbSGT1 further exacerbated hyphal spread, suggesting synergistic contributions of SA signaling and SGT1-dependent ETI to NHR.
Although classical quantitative methods such as electrolyte leakage assays and fungal biomass measurements are commonly employed to evaluate plant-pathogen interactions, their application was limited in this study. Despite inoculating N. benthamiana leaves with a high concentration of M. oryzae conidia, observable infection events remained rare, and HR-like cell death phenotypes became apparent only at late stages (8 dpi), in contrast to the rapid HR triggered in rice at 2 dpi. This reflects the fundamentally low infection efficiency of M. oryzae in the nonhost N. benthamiana. Nonetheless, detailed microscopic observations consistently revealed hallmark features of HR, including localized brown discoloration confined to initially invaded cells, restricted hyphal expansion, and delayed fungal development. These phenotypic observations, in combination with the upregulation of defense marker genes such as NbPR1, NbWRKY22, and NbCYP71D20, strongly support the activation of PTI- and ETI-like immune responses. Thus, while quantitative measurements were not feasible under the current experimental conditions, the convergence of cytological and transcriptional evidence provides a robust basis for our conclusions.
Robust NLR-mediated immunity in nonhost plants: uncovering SGT1-dependent ETI as a blueprint for durable resistance
High-throughput screening of 179 candidate MOEs using a PVX-based expression system revealed that 70 MOEs triggered HR cell death in N. benthamiana, indicating the activation of ETI through effector recognition. Further analysis demonstrated that silencing of NbSGT1 abolished MOE-induced HR cell death, underscoring the essential role of SGT1-dependent NLR signaling in nonhost defense. These findings suggest that N. benthamiana maintains a diverse NLR repertoire capable of recognizing a broad range of non-adapted pathogen effectors. Consistent with previous studies in pepper (CaRpi-blb2 against Phytophthora infestans), rice (engineered Pikp NLRs recognizing expanded AVR-Pik variants), and Arabidopsis (ZAR1-ZRK3 complex-mediated recognition), our results highlight the remarkable plasticity of NLR-mediated immunity across plant species. Although it has been proposed that increasing phylogenetic divergence between host and pathogen shifts NHR reliance from ETI to PTI (Schulze-Lefert and Panstruga, 2011), our findings challenge this view by demonstrating that ETI, mediated by SGT1 and SA, remains critical for blocking M. oryzae colonization in the distantly related dicot N. benthamiana.
Delayed appressorium formation and conidial abnormalities in nonhost interactions
While the overall infection cycle of M. oryzae was initiated in N. benthamiana, important differences compared to susceptible rice interactions were observed. In rice, penetration peg formation, IH expansion, and HR occur rapidly by 2 dpi, while in N. benthamiana, localized cell death developed only after 6-8 dpi. Fungal entry was also rare, suggesting impaired early infection stages. This delay may be attributed to the absence of host-specific cues required for appressorium maturation and penetration, as well as intrinsic differences in cell wall composition and surface properties between monocots and dicots. Additionally, conidial abnormalities, such as shrinkage observed in EV, NbEAS-, and NbBAK1-silenced N. benthamiana plants, may result from exposure to antimicrobial compounds, independent of capsidiol. These abnormalities parallel findings from treatments with antimicrobial peptides such as EcAMP1 (Vasilchenko et al., 2016), suggesting a contribution of chemical barriers to NHR.
Conclusion
This study provides new insights into the mechanisms underlying NHR in N. benthamiana against the blast fungus M. oryzae. Our findings demonstrate that SGT1 and SA orchestrate robust ETI and HR cell death, restricting fungal invasion. The high frequency of HR triggered by MOEs suggests that nonhost plants harbor a broad NLR repertoire capable of recognizing diverse effectors from unadapted pathogens. Moreover, delayed infection dynamics, restricted hyphal expansion, and conidial abnormalities further highlight the multi-layered nature of NHR. These results underscore the potential of exploiting nonhost NLRs and immune regulators such as SGT1 to engineer durable, broad-spectrum resistance against rapidly evolving pathogens in crops.
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