Biology subjects

Khang, C. H.

Publications and source records attributed to Khang, C. H..

7 recordsLinked to original sources

bioRxiv2026

In planta-specific transcriptional regulatory circuit regulates expression of MoHTR1, a nuclear effector gene of Magnaporthe oryzae

During host-pathogen interactions, fungal pathogens secrete effector proteins into host cells to manipulate the host immune system and facilitate infection. Although many effector genes are highly expressed during infection stages, there is limited information on the mechanisms regulating their in planta expression. Here, we characterize the promoter of MoHTR1, a nuclear effector gene of the rice blast fungal pathogen, to elucidate its in planta-specific expression. Using promoter deletion and mutation analyses, we identified a core cis-element (TATTTCGT) within the MoHTR1 promoter, designated the in planta active (IPA) element, which is crucial for in planta-specific expression. The IPA element is responsible for the expression of not only MoHTR1, but also other effector genes including a known effector Slp1. Furthermore, the IPA element enables the in planta expression of MobZIP14, a gene specifically expressed during vegetative growth. The IPA element plays a critical role in fungal virulence by enabling MoHTR1 expression and regulating host immune responses. Bioinformatic and DNA-protein interaction analyses revealed that RGS1, a transcription factor containing a winged-helix binding domain, acts as a transcriptional regulator of MoHTR1 by directly binding to the IPA element. Our findings provide new insights into the regulatory mechanisms driving the in planta-specific expression of fungal effector genes.

microbiology↗

bioRxiv2025

Pan-genome analyses of 226 finger millet-infecting Magnaporthe oryzae strains from eastern Africa reveal two lineages that differ in their genetic make-up, virulence and effector gene content with gene loss driven by homologous recombination between Pot2 elements

Blast disease, caused by the filamentous ascomycete Magnaporthe oryzae, is the main biotic constraint to finger millet production in eastern Africa. M. oryzae is a pathogen on many grass species, but the high host-specificity of blast isolates underscores the need to study pathogen diversity and virulence at the host level. Here, we fill a void on knowledge on finger millet-infecting strains of M. oryzae by sequencing 226 isolates pure-cultured from infected tissues, mainly peduncles and panicles, sampled from field-grown finger millet accessions across Ethiopia, Kenya, Tanzania and Uganda. Phylogenetic analysis showed that eastern African isolates are genetically distinct from Asian isolates, and differentiated into two groups potentially driven by climate variables. One group was dominated by isolates from Kenya and Uganda (KU group), and the other by isolates from Ethiopia and Tanzania (ET group). Analysis of the portfolio of 887 predicted effector genes showed that the KU group had significantly fewer effectors, concomitant with higher virulence levels on the two finger millet accessions tested. We demonstrate that homologous recombination between transposable elements, leading to genomic deletions, plays a key role in gene removal. Transcript profiling of fungal genes in compatible and incompatible interactions revealed upregulation in the incompatible interaction as a characteristic of an estimated 30% of effector genes, including seven of the eight homologs of known avirulence genes from rice-infecting M. oryzae identified in our study. Prioritization of these effectors for functional validation will pave the way for identifying cognate resistance genes and improving finger millet for resistance to blast disease.

plant biology↗

bioRxiv2021

The mitotic spindle mediates nuclear migration through an extremely narrow infection structure of the rice blast fungus Magnaporthe oryzae

The blast fungus, Magnaporthe oryzae, causes severe destruction to rice and other crops worldwide. As the fungus infects rice, it develops unique cellular structures, such as an appressorium and a narrow penetration peg, to permit successful invasion of host rice cells. Fundamental knowledge about these cellular structures and how organelles, such as the nucleus, are positioned within them is still emerging. Previous studies show that a single nucleus becomes highly stretched during movement through the narrow penetration peg in an extreme nuclear migration event. Yet, the mechanism permitting this nuclear migration event remains elusive. Here, we investigate the role of the mitotic spindle in mediating nuclear migration through the penetration peg. We find that disruption of spindle function during nuclear migration through the penetration peg prevents development of invasive hyphae and virulence on rice. Furthermore, regulated expression of conserved kinesin motor proteins, MoKin5 and MoKin14, is essential to form and maintain the spindle, as well as, properly nucleate the primary hypha. Overexpression of MoKin5 leads to formation of aberrant microtubule protrusions, which contributes to formation of nuclear fragments within the appressorium and primary hypha. Conversely, overexpression of MoKin14 causes the spindle to collapse leading to the formation of monopolar spindles. These results establish a mechanistic model towards understanding the intricate subcellular dynamics of extreme nuclear migration through the penetration peg, a critical step in the development of rice blast disease. ImportanceMagnaporthe oryzae, also known as the blast fungus, is a formidable hinderance to global food production, including rice. The destructive fungal pathogen develops highly-specialized cells and structures, such as appressoria and penetration pegs, to permit successful invasion of rice cells. Our understanding of M. oryzaes fundamental biology during host cell invasion and colonization is still developing. For instance, it is not yet known how organelles, such as the nucleus, migrate through the narrow penetration peg. Moreover, few previous studies examine the role of motor proteins in M. oryzae. In this study, we determined that the mitotic spindle propels a single nucleus through the penetration peg to permit successful development of fungal hyphae inside the first-invaded rice cell. We also identified two conserved kinesin motor proteins, MoKin5 and MoKin14. Our analyses suggested that MoKin5 and MoKin14 exhibit canonical functions in M. oryzae during rice infection. This study addressed long-standing questions in rice blast biology, and our results offer opportunities for future research.

cell biology↗

bioRxiv2020

Role of two metacaspases in development and pathogenicity of the Rice Blast fungus, Magnaporthe oryzae

Rice blast disease caused by Magnaporthe oryzae is a devastating disease of cultivated rice worldwide. Infections by this fungus lead to a significant reduction in rice yields and threats to food security. To gain better insight into growth and cell death in M. oryzae during infection, we characterized two predicted M. oryzae metacaspase proteins, MoMca1 and MoMca2. These proteins appear to be functionally redundant and are able to complement the yeast Yca1 homologue. Biochemical analysis revealed that M. oryzae metacaspases exhibited Ca2+ dependent caspase activity in vitro. Deletion of both MoMca1 and MoMca2 in M. oryzae resulted in reduced sporulation, delay in conidial germination and attenuation of disease severity. In addition, the double {Delta}Momca1mca2 mutant strain showed increased radial growth in the presence of oxidative stress. Interestingly, the {Delta}Momca1mca2 strain showed an increase accumulation of insoluble aggregates compared to the wild-type strain during vegetative growth. Our findings suggest that MoMca1 and MoMca2 promote the clearance of insoluble aggregates in M. oryzae, demonstrating the important role these metacaspases have in fungal protein homeostasis. Furthermore, these metacaspase proteins may play additional roles, like in regulating stress responses, that would help maintain the fitness of fungal cells required for host infection. IMPORTANCEMagnaporthe oryzae causes rice blast disease that threatens global food security by resulting in the severe loss of rice production every year. A tightly regulated life cycle allows M. oryzae to disarm the host plant immune system during its biotrophic stage before triggering plant cell death in its necrotrophic stage. The ways M. oryzae navigates its complex life cycle remains unclear. This work characterizes two metacaspase proteins with peptidase activity in M. oryzae that are shown to be involved in the regulation of fungal growth and development prior to infection by potentially helping maintain fungal fitness. This study provides new insight into the role of metacaspase proteins in filamentous fungi by illustrating the delays in M. oryzae morphogenesis in the absence of these proteins. Understanding the mechanisms by which M. oryzae morphology and development promote its devastating pathogenicity may lead to the emergence of proper methods for disease control.

microbiology↗

bioRxiv2020

Full three-dimensional imaging deep through multicellular thick samples with subcellular resolution by structured illumination microscopy and adaptive optics

Structured Illumination Microscopy enables live imaging with resolutions of ~120 nm. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 140 nm lateral and 585 nm axial resolution in tissue culture cells, C. elegans, and rice blast fungus. We demonstrate that AO improves resolution and reduces artifacts, making full 3D SIM possible in thicker samples.

cell biology↗

bioRxiv2020

Pantoea ananatis defeats Allium chemical defenses with a plasmid-borne virulence gene cluster

Onion (Allium. cepa L), garlic (A. sativum L.), and other members of the Allium genus produce volatile antimicrobial thiosulfinates upon cellular damage. Allicin has been known since the 1950s as the primary antimicrobial thiosulfinate compound and odorant produced by garlic. However, the roles of endogenous thiosulfinate production in host-bacterial pathogen interactions have not been described. The bacterial onion pathogen Pantoea ananatis, which lacks both the virulence Type III and Type II Secretion Systems, induces necrotic symptoms and extensive cell death in onion tissues dependent on a proposed secondary metabolite synthesis chromosomal gene cluster. We found strong correlation between the genetic requirements for P. ananatis to colonize necrotized onion tissue and its capacity for tolerance to the thiosulfinate allicin based on the presence of an eleven gene, plasmid-borne, virulence cluster of sulfur/redox genes. We have designated them alt genes for allicin tolerance. We show that allicin and onion thiosulfinates restrict bacterial growth with similar kinetics. The alt gene cluster is sufficient to confer allicin tolerance and protects the glutathione pool during allicin treatment. Independent alt genes make partial phenotypic contributions indicating that they function as a collective cohort to manage thiol stress. Our work implicates endogenous onion thiosulfinates produced during cellular damage as mediators of interactions with bacteria. The P. ananatis-onion pathosystem can be modeled as a chemical arms race of pathogen attack, host chemical counter-attack, and pathogen resistance. Significance StatementAlliums (e.g. onion and garlic), after sustaining cellular damage, produce potent antimicrobial thiosulfinates that react with cellular thiols. The bacterial onion pathogen Pantoea ananatis, which lacks the virulence Type III and Type II Secretion Systems, induces cell death and necrotic symptoms on onions. We have identified a plasmid-borne cluster of sulfur/redox virulence genes that 1) are required for P. ananatis to colonize necrotized onion tissue, 2) are sufficient for tolerance to the thiosulfinates, and, 3) protect the glutathione pool during thiosulfinate treatment. We propose that the thiosulfinate production potential of Allium spp. governs Allium-bacterial interaction outcomes and that the P. ananatis-onion pathosystem can be modeled as a chemical arms race of attack and counterattack between the pathogen and host.

plant biology↗

bioRxiv2017

Disruption of the interfacial membrane leads to Magnaporthe oryzae effector re-location and lifestyle switch during rice blast disease

The hemibiotrophic fungus Magnaporthe oryzae produces invasive hyphae enclosed in a plant-derived interfacial membrane, known as the extra-invasive hyphal membrane (EIHM), in living rice cells. Little is known about when the EIHM is disrupted and how the disruption contributes to blast disease. Here we show that EIHM disruption correlates with the hyphal growth stage in first-invaded susceptible rice cells. Our approach utilized GFP secreted from invasive hyphae as an EIHM integrity reporter. Secreted-GFP accumulated in the EIHM compartment but appeared in the rice cytoplasm when the EIHM integrity was compromised. Live-cell imaging of secreted-GFP and various fluorescent reporters revealed that EIHM disruption led to rice vacuole rupture and cell death limited to the invaded cell with closed plasmodesmata. We report that EIHM disruption and host cell death are landmarks delineating three distinct infection phases (early biotrophic, late biotrophic, and transient necrotrophic phases) within the first-invaded cell before reestablishment of biotrophy in second-invaded cells. M. oryzae effectors exhibited phase-specific localizations, including entry of the apoplastic effector Bas4 into the rice cytoplasm during the late biotrophic phase. Understanding how the phase-specific dynamics are regulated and linked to host susceptibility will offer potential targets that can be exploited to control blast disease.

cell biology↗