Biology subjects

Sung, Y.-C.

Publications and source records attributed to Sung, Y.-C..

7 recordsLinked to original sources

bioRxiv2026

Spatial organization of plant defense at the infection front

Plants lack specialized immune cells and instead rely on coordinated cellular responses to restrict pathogen invasion while preserving tissue integrity. How plant immunity spatially organizes these responses remains unclear. Using live-cell reporters in Arabidopsis infected with Pseudomonas syringae, we show that effector-triggered immunity, superimposed on pattern-triggered immunity, establishes a sustained yet spatially confined immune architecture at the infection front. Defense activation persists for days but remains restricted to a narrow ring of cells surrounding viable bacterial microcolonies. Over time, immune activity spreads to adjacent layers, forming a coordinated, multilayered defense zone. This zonation extends beyond transcriptional activation to polarized callose deposition at pathogen-facing cell walls, reinforcing a localized containment boundary that limits pathogen spread. Consistent with previous single-reporter studies, simultaneous visualization of salicylic acid (SA) and jasmonic acid (JA) biosynthesis and response markers reveals a radial hormone gradient, with SA-enriched cells proximal to bacterial colonies and JA-enriched cells in surrounding regions. Individual cells predominantly activate one pathway, indicating that SA-JA antagonism is resolved through spatial compartmentalization across neighboring cells. Together, these findings establish immune zonation as a strategy for robust pathogen containment while minimizing collateral tissue damage. Significance statementPlants lack specialized immune cells and must coordinate defense responses across tissues to contain infection. Although plant immune responses are known to be locally activated, how they are organized across cells to form a stable containment boundary has remained unclear. Using live imaging of fluorescent bacterial microcolonies, we show that plant immunity forms a spatially organized defense zone surrounding viable infection sites. Within this zone, distinct defense activities are arranged in a radial gradient, with salicylic acid-associated responses enriched in cells near the pathogen and jasmonic acid-associated responses in surrounding cells. Rather than being resolved solely within individual cells, antagonistic hormone pathways are separated across neighboring cells. This spatial organization reveals immune zonation as a strategy for confining pathogen spread while preserving tissue integrity.

plant biology↗

bioRxiv2024

Revisiting the evolution and function of NIP2 parologs in the Rhynchosporium spp. complex

The fungus Rhynchosporium commune, the causal agent of barley scald disease, contains a paralogous effector gene family called Necrosis-Inducing Protein 2 (NIP2) and NIP2-like protein (NLP). However, the function and full genomic context of these paralogs remains uncharacterised. Here we present a highly contiguous long-read assembly of R. commune WAI453. Using this assembly, we show that the duplication of the NIP2 and NLP gene families is distributed throughout the genome and pre-dates the speciation of R. commune from its sister species. Some NIP2 paralogs have subsequently been lost or are absent in the sister species. The diversity of these paralogs was examined from R. commune global populations and their expression was analysed during in planta and in vitro growth to analyse the importance of these genes during infection. The majority of NIP2 and NLP paralogs in WAI453 genome were significantly upregulated during plant infection suggesting that the NIP2 and NLP genes harbour virulence roles. An attempt to further characterise function of NIP2.1 by infiltrating purified protein into barley leaves did not induce necrosis questioning its previously reported role as an inducer of host cell death. Together these results suggest that the NIP2 effector family does play a role during infection of barley, however the exact function of NIP2, like many effectors, remains uncharacterised.

plant biology↗

bioRxiv2024

Direct binding of a fungal effector by the wheat RWT4 tandem kinase activates defense

Plants have intricate innate immune receptors that detect pathogens. Research has intensely focused on two receptor classes recognizing external and internal threats. Recent research has identified a class of disease resistance proteins called tandem kinase proteins (TKPs). We investigated RWT4, a wheat TKP that confers resistance to the devastating fungal pathogen Magnaporthe oryzae. We established a rice protoplast system, revealing RWT4 specifically recognizes the AvrPWT4 effector, leading to the transcription of defense genes and inducing cell death. RWT4 possesses both kinase and pseudokinase domains, with its kinase activity essential for defense. RWT4 directly interacts with and transphosphorylates AvrPWT4. Biolayer interferometry revealed both RWT4 kinase and pseudokinase regions bind the effector. Sequence similarity and structural modeling revealed an integrated partial kinase duplication in RWT4s kinase region as critical for effector interaction and defense activation. Collectively, these findings demonstrate that TKPs can directly bind a recognized effector, leading to downstream defense activation.

plant biology↗

bioRxiv2022

Membrane Protein Modification Modulates Big and Small Extracellular Vesicle Biodistribution and Tumorigenic Potential in Breast Cancers In Vivo

Extracellular vesicles (EVs) are released by cells to mediate intercellular communication under pathological and physiological conditions. While small EVs (sEVs; <100-200 nm, exosomes) are intensely investigated, the properties and functions of medium and large EVs (big EVs [bEVs]; >200 nm, microvesicles) are less well explored. Here, we identify bEVs and sEVs as distinct EV populations, and determine that bEVs are released in a greater bEV:sEV ratio in the aggressive human triple-negative breast cancer (TNBC) subtype. PalmGRET, bioluminescence resonance energy transfer (BRET)-based EV reporter, reveals dose- dependent EV biodistribution at non-lethal and physiological EV dosages, as compared to lipophilic fluorescent dyes. Remarkably, the bEVs and sEVs exhibit unique biodistribution profiles, et individually promote in vivo tumor growth in a syngeneic immunocompetent TNBC breast tumor murine model. The bEVs and sEVs share mass spectrometry (MS)- identified tumor progression-associated EV surface membrane proteins (tpEVSurfMEMs), which include SLC29A1, CD9 and CD44. tpEVSurfMEM depletion attenuates EV lung organotropism, alters biodistribution, and reduces protumorigenic potential. This study identifies distinct in vivo property and function of bEVs and sEVs in breast cancer, which suggest the significant role of bEVs in diseases, diagnostic and therapeutic applications.

cancer biology↗

bioRxiv2021

Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be useful when investigating the function and recognition of a broad range of disulfide-bond containing effectors.

biochemistry↗

bioRxiv2020

The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and insights into Kex2 protease processing of fungal effectors

O_LIPlant pathogens cause disease through secreted effector proteins, which act to modulate host physiology and promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilised a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3. C_LIO_LIWe developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination. We show this approach can be successfully applied to effectors from other pathogenic fungi. Complementing this, an in-silico study uncovered the prevalence of an expanded subclass of effectors from fungi. C_LIO_LIThe {beta}-barrel fold of SnTox3 is a novel fold among fungal effectors. We demonstrate that SnTox3 is a pre-pro-protein and that the protease Kex2 removes the pro-domain. Our in-silico studies suggest that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi, and we demonstrate this experimentally for effectors from Fusarium oxysporum f sp. lycopersici. C_LIO_LIWe propose that K2PP effectors are highly prevalent among fungal effectors. The identification and classification of K2PP effectors has broad implications for the approaches used to study their function in fungal virulence. C_LI

plant biology↗

bioRxiv2020

Redirected tropisms of extracellular vesicles and exomeres yield distinct biodistribution profiles

Extracellular particles (EP) including extracellular vesicles (EVs) and exomeres have been shown to play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. We therefore created a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, to enable pan-EP labelling ranging from exomeres (< 50 nm) to small (< 200 nm) and medium and large (> 200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking and quantification of the EPs from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET and fluorescence. Using PalmGRET, we show that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. We further demonstrate that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1 (Slco2a1), alanine aminopeptidase (Cd13) and chloride intracellular channel (Clic1) decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. We anticipate that EP-specific imaging, quantitative assays and detailed in vivo characterization to be a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.

cell biology↗