Rice floret infection strategy unraveled

Abstract

Ustilaginoidea virens, the causal agent of false smut in rice, poses a significant threat to global rice production, with yield losses of up to 53% reported in India (Masurkar et al., 2023) and increasing incidence world-wide (Adhikari, 2024). This ascomycete employs an intriguing plant infection strategy by colonizing rice florets through the stamens of the florets, apparently without the use of appressoria or other visible infection structures. Notably, it may bypass conventional plant penetration mechanisms depending on cell wall degradation, as its genome harbors relatively few genes encoding cell-wall degrading enzymes compared to other phytopathogenic ascomycetes (Zhang et al., 2014). In an article recently published in New Phytologist, Yu et al. (2025; doi: 10.1111/nph.70414) now find that U. virens secretes a small effector protein into the host tissue. This effector interacts with a plant target protein to release two MADS transcription factors from the chloroplast into the nucleus, where they activate gibberellin (GA) biosynthesis. Elevated GA levels promote plant elongation, suppress immune responses, and induce the expression of expansins. Expansins are proteins that lead to cell-wall loosening (Cosgrove, 2024). Together, these effects likely facilitate hyphal invasion of the stamens by weakening physical barriers and dampening plant defenses.

This study stands out as a textbook example of effector function elucidation through a combination of rigorous experimentation and hypothesis-driven exploration…

Yu et al. started with the quest to uncover the role of predicted secreted cysteine-rich effectors (SCREs) that were discovered after genome sequencing (Zhang et al., 2014). Among these, UvSCRE9 – a 142-amino acid protein lacking known conserved domains but containing three conserved motifs of unknown function – is highly upregulated during plant infection. They first validated the functionality of its predicted secretion signal peptide by two heterologous secretion assays. In Saccharomyces cerevisiae, the UvSCRE9 signal peptide directed secretion of an invertase lacking its native signal peptide to the culture medium. In Magnaporthe oryzae, the effector directed the green fluorescent protein (GFP) to the biotrophic interfacial complex, a plant-derived membrane-rich structure that is targeted by some proven plant-transferred M. oryzae effector proteins (Oliveira-Garcia et al., 2024). Deletion of UvSCRE9 from the genome of U. virens severely reduced its ability to cause false smut symptoms in rice. This virulence defect was rescued by overexpression of a FLAG-tagged UvSCRE9 transgene in the mutant background, confirming the effector's important role in pathogenicity.

To investigate the effect of UvSCRE9 on host immunity, Yu et al. generated transgenic rice lines expressing the effector under a dexamethasone (DEX)-inducible promoter. Upon DEX treatment, these lines exhibited significantly reduced oxidative bursts in response to chitin and flg22, compared to both untreated transgenic lines and wild-type controls. This suppression of pattern-triggered immunity (PTI) was further supported by the attenuated induction of defense marker genes PR10 and PR1b in DEX-treated transgenic plants. These results demonstrate that UvSCRE9, a secreted virulence effector of U. virens, effectively dampens PTI responses in rice.

In parallel, the authors generated transgenic rice lines constitutively expressing FLAG-tagged UvSCRE9. These plants showed increased height and morphological changes in root tip cells, including elongation and loosening – traits suggestive of altered cell wall dynamics.

To explore the underlying mechanisms of UvSCRE9 function, the researchers analyzed the transcriptome of wild-type rice panicles and panicles from DEX-treated UvSCRE9-expressing lines. Approximately 600 genes were upregulated in response to effector expression, many of which are involved in cell wall modification. Notably, genes encoding expansins, pollen allergens, and β-glucosidases were dramatically upregulated. While β-glucosidases contribute to cellulose degradation, expansins and pollen allergens facilitate cell-wall loosening – particularly in stamen filaments, the key entry site for U. virens hyphae. These findings suggest that UvSCRE9 reprograms host gene expression to promote tissue conditions favorable for fungal colonization.

Among the UvSCRE9-upregulated plant genes, Yu et al. identified several associated with GA biosynthesis. Accordingly, they observed a significant increase in active GAs in UvSCRE9 lines.

To assess the functional relevance of this increase, they manipulated GA levels before U. virens inoculation: exogenous GA markedly enhanced the incidence of false-smut balls, whereas the application of a GA-biosynthesis inhibitor greatly suppressed disease symptoms. Furthermore, dexamethasone-induced UvSCRE9 increased susceptibility, and the combination of UvSCRE9 with GA treatment produced an exceptionally severe disease phenotype. Together, these results confirm that both GA accumulation and UvSCRE9 are decisive for U. virens virulence in rice.

To determine whether UvSCRE9 and GA act independently, the authors evaluated susceptibility to the bacterial pathogen Xanthomonas oryzae pv oryzae. While GA treatment increased lesion size and UvSCRE9 further exacerbated symptoms, the inhibition of GA biosynthesis reduced lesion sizes to the same low level regardless of UvSCRE9. This demonstrates that UvSCRE9, rather than functioning independently, promotes virulence by activating GA biosynthesis.

To investigate how UvSCRE9 influences GA signaling, Yu et al. used their rice lines that constitutively expressed FLAG-tagged UvSCRE9 for immunoprecipitation followed by mass spectrometry to identify host interaction partners. Among the candidates, they identified one plant protein (SCRE9-interacting protein 1; OsSIP1) whose interaction could be validated by both yeast two-hybrid and co-immunoprecipitation experiments.

Because OsSIP1 was uncharacterized, they hypothesized that it might associate with floral regulators of GA biosynthesis, specifically OsMADS63 and OsMADS68. Indeed, immunoprecipitation and bimolecular fluorescence complementation demonstrated that OsSIP1 interacts with both transcription factors.

Next, they tested whether OsMADS63 and OsMADS68 directly regulate GA biosynthesis in rice. They found two putative MADS transcription factor binding sites in the promoter of the OsGA3ox1 gene, coding for an enzyme catalyzing the rate-limiting step in GA biosynthesis. Electrophoretic mobility shift assays confirmed the binding of OsMADS63 and OsMADS68 to these sites. Using a GA3ox1 promoter-luciferase reporter construct in rice protoplasts, the researchers further showed that OsMADS63 and OsMADS68 activate GA3ox1 expression, while OsSIP1 represses it. UvSCRE9 alone did not activate GA3ox1, but its presence counteracted the repressive effect of OsSIP1. This indicates that UvSCRE9 interferes with OsSIP1, which normally suppresses OsMADS63/68-mediated activation of GA3ox1.

Yu et al. then examined the subcellular localization of the four proteins by expressing GFP- and HA-tagged fusion constructs in rice protoplasts and Nicotiana benthamiana leaves. When expressed individually, OsSIP1 was found in chloroplasts, OsMADS63/68 in both the nucleus and cytoplasm, and UvSCRE9 in the cytosol, nucleus and chloroplasts. Co-expression, however, altered these localization patterns: in the presence of UvSCRE9, OsSIP1 shifted from chloroplasts to the nucleus and cytoplasm, while the co-expression of OsSIP1 with its interacting transcription factors recruited OsMADS63/68 into chloroplasts. Importantly, chloroplast recruitment of OsMADS63/68 by OsSIP1 was strongly reduced when UvSCRE9 was present.

The findings now provide a clear model of how a small secreted fungal effector protein hijacks GA biosynthesis in rice. Under normal conditions, OsMADS63 and OsMADS68 are sequestered in chloroplasts via OsSIP1 binding, thereby limiting their ability to activate transcription. During fungal colonization, UvSCRE9 binds to OsSIP1, causing its relocalization to the nucleus and cytoplasm. This, in turn, frees OsMADS63/68 to accumulate in the nucleus where they activate GA3ox1 transcription, driving production of the rate-limiting enzyme in GA biosynthesis. The resulting accumulation of active GAs suppresses PTI while promoting expression of expansins and pollen allergens – factors that facilitate U. virens colonization of rice stamens (Fig. 1).

Fig. 1

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Mechanistic model of UvSCRE9 function in the Ustilaginoidea virens–rice interaction. (Left) In the absence of fungal infection, OsSIP1 (green circle with triangle cut out) interacts with the transcription factors OsMADS63/68 (blue spiked rectangle), sequestering them in the chloroplasts and preventing gibberellin (GA) biosynthesis. (Right) During infection, U. virens secretes the small cysteine-rich effector UvSCRE9 (auburn triangle) that binds OsSIP1 in the cytoplasm and the nucleus. This interaction disrupts OsSIP1 function and allows OsMADS63/68 to relocate to the nucleus, where they activate GA3ox1 transcription, encoding the enzyme and catalyzing the rate-limiting step in GA biosynthesis. Elevated GA levels trigger the expression of expansins (black rectangles), pollen antigens, and β-glucosidases, thereby suppressing pattern-triggered immunity and loosening cell walls to facilitate the spread of U. virens hyphae.

This study stands out as a textbook example of effector function elucidation through a combination of rigorous experimentation and hypothesis-driven exploration, offering a methodological blueprint for researchers investigating effectors in diverse pathosystems. Moreover, it identifies the first effector known to directly manipulate GA signaling, thereby firmly establishing a link between GA signaling and plant immunity. I therefore foresee that UvSCRE9 will not remain the only effector targeting this hormone pathway – other effectors and other pathosystems will be discovered that exploit GA signaling to promote host plant colonization.