Chang Hyun Khang

Translocation of Magnaporthe oryzae Effectors into Rice Cells and Their Subsequent Cell-to-Cell Movement

The authors imaged fungal transformants secreting fluorescent effector fusion proteins in first-invaded rice cells. Two effectors that accumulated in biotrophic interfacial complexes were translocated into the invaded cells cytoplasm. Depending on rice cell type and effector size, the translocated effectors moved into adjoining uninvaded rice cells, potentially preparing them for fungal entry. Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice.

Interaction Transcriptome Analysis Identifies Magnaporthe oryzae BAS1-4 as Biotrophy-Associated Secreted Proteins in Rice Blast Disease

Biotrophic invasive hyphae (IH) of the blast fungus Magnaporthe oryzae secrete effectors to alter host defenses and cellular processes as they successively invade living rice (Oryza sativa) cells. However, few blast effectors have been identified. Indeed, understanding fungal and rice genes contributing to biotrophic invasion has been difficult because so few plant cells have encountered IH at the earliest infection stages. We developed a robust procedure for isolating infected-rice sheath RNAs in which ∼20% of the RNA originated from IH in first-invaded cells. We analyzed these IH RNAs relative to control mycelial RNAs using M. oryzae oligoarrays. With a 10-fold differential expression threshold, we identified known effector PWL2 and 58 candidate effectors. Four of these candidates were confirmed to be fungal biotrophy-associated secreted (BAS) proteins. Fluorescently labeled BAS proteins were secreted into rice cells in distinct patterns in compatible, but not in incompatible, interactions. BAS1 and BAS2 proteins preferentially accumulated in biotrophic interfacial complexes along with known avirulence effectors, BAS3 showed additional localization near cell wall crossing points, and BAS4 uniformly outlined growing IH. Analysis of the same infected-tissue RNAs with rice oligoarrays identified putative effector-induced rice susceptibility genes, which are highly enriched for sensor-transduction components rather than typically identified defense response genes.

Genome-wide functional analysis of pathogenicity genes in the rice blast fungus.

Rapid translation of genome sequences into meaningful biological information hinges on the integration of multiple experimental and informatics methods into a cohesive platform. Despite the explosion in the number of genome sequences available, such a platform does not exist for filamentous fungi. Here we present the development and application of a functional genomics and informatics platform for a model plant pathogenic fungus, Magnaporthe oryzae. In total, we produced 21,070 mutants through large-scale insertional mutagenesis using Agrobacterium tumefaciens–mediated transformation. We used a high-throughput phenotype screening pipeline to detect disruption of seven phenotypes encompassing the fungal life cycle and identified the mutated gene and the nature of mutation for each mutant. Comparative analysis of phenotypes and genotypes of the mutants uncovered 202 new pathogenicity loci. Our findings demonstrate the effectiveness of our platform and provide new insights on the molecular basis of fungal pathogenesis. Our approach promises comprehensive functional genomics in filamentous fungi and beyond.

The Magnaporthe oryzae Effector AvrPiz-t Targets the RING E3 Ubiquitin Ligase APIP6 to Suppress Pathogen-Associated Molecular Pattern-Triggered Immunity in Rice

This work shows that the Magnaporthe oryzae effector AvrPiz-t enters into rice cells to target the RING E3 ubiquitin ligase APIP6 for suppression of PAMP-triggered immunity in rice. It also describes that APIP6 degrades AvrPiz-t in planta and positively regulates basal defense to M. oryzae. Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.

Recent advances in rice blast effector research.

To cause rice blast disease, the fungus Magnaporthe oryzae produces biotrophic invasive hyphae that secrete effectors at the host-pathogen interface. Effectors facilitate disease development, but some (avirulence effectors) also trigger the hosts resistance gene-mediated hypersensitive response and block disease. The number of cloned M. oryzae avirulence effector genes has recently doubled, largely based on resequencing with a Japanese field isolate and association of avirulence activity with presence/absence polymorphisms in novel genes for secreted proteins. Effectors secreted by hyphae in rice cells accumulate in biotrophic interfacial complexes, and this property correlates with their translocation across plasma membrane into the rice cytoplasm. Interestingly, the translocated effectors moved into surrounding uninvaded cells, suggesting that effectors prepare host cells before the fungus enters them.

Genome Organization and Evolution of the AVR-Pita Avirulence Gene Family in the Magnaporthe grisea Species Complex

The avirulence (AVR) gene AVR-Pita in Magnaporthe oryzae prevents the fungus from infecting rice cultivars containing the resistance gene Pi-ta. A survey of isolates of the M. grisea species complex from diverse hosts showed that AVR-Pita is a member of a gene family, which led us to rename it to AVR-Pita1. Avirulence function, distribution, and genomic context of two other members, named AVR-Pita2 and AVR-Pita3, were characterized. AVR-Pita2, but not AVR-Pita3, was functional as an AVR gene corresponding to Pi-ta. The AVR-Pita1 and AVR-Pita2 genes were present in isolates of both M. oryzae and M. grisea, whereas the AVR-Pita3 gene was present only in isolates of M. oryzae. Orthologues of members of the AVR-Pita family could not be found in any fungal species sequenced to date, suggesting that the gene family may be unique to the M. grisea species complex. The genomic context of its members was analyzed in eight strains. The AVR-Pita1 and AVR-Pita2 genes in some isolates appeared to be located near telomeres and flanked by diverse repetitive DNA elements, suggesting that frequent deletion or amplification of these genes within the M. grisea species complex might have resulted from recombination mediated by repetitive DNA elements.

The ER chaperone LHS1 is involved in asexual development and rice infection by the blast fungus Magnaporthe oryzae.

In planta secretion of fungal pathogen proteins, including effectors destined for the plant cell cytoplasm, is critical for disease progression. However, little is known about the endoplasmic reticulum (ER) secretion mechanisms used by these pathogens. To determine if normal ER function is crucial for fungal pathogenicity, Magnaporthe oryzae genes encoding proteins homologous to yeast Lhs1p and Kar2p, members of the heat shock protein 70 family in Saccharomyces cerevisiae, were cloned and characterized. Like their yeast counterparts, both LHS1 and KAR2 proteins localized in the ER and functioned in an unfolded protein response (UPR) similar to the yeast UPR. Mutants produced by disruption of LHS1 were viable but showed a defect in the translocation of proteins across the ER membrane and reduced activities of extracellular enzymes. The Δlhs1 mutant was severely impaired not only in conidiation, but also in both penetration and biotrophic invasion in susceptible rice (Oryza sativa) plants. This mutant also had defects in the induction of the Pi-ta resistance gene–mediated hypersensitive response and in the accumulation of fluorescently-labeled secreted effector proteins in biotrophic interfacial complexes. Our results suggest that proper processing of secreted proteins, including effectors, by chaperones in the ER is requisite for successful disease development and for determining host-pathogen compatibility via the gene-for-gene interaction.

Filamentous Fungi (Magnaporthe grisea and Fusarium oxysporum).

A better understanding of fungal biology will facilitate judicious use of beneficial fungi and will also advance our efforts to control pathogenic fungi. Molecular studies of fungal biology have been greatly aided by transformation-mediated mutagenesis techniques. Transformation via nonhomologous integration of plasmid DNA bearing a selectable marker (e.g., antibiotic resistance gene) has been widely used for the random insertional mutagenesis of fungi - as an alternative to chemical and radiation mutagens - mainly because the integration of plasmid into the genome provides a convenient tag for subsequent identification and isolation of the mutated gene. Homologous recombination between a target gene on the chromosome and the introduced DNA carrying its mutant allele results in targeted gene knock-out. An important advance in fungal transformation methodology is the development of Agrobacterium tumefaciens-mediated transformation (ATMT) protocols for fungi. ATMT has been successfully applied to a phylogenetically diverse group of fungi and offers a number of advantages over conventional transformation techniques in both the random insertional mutagenesis and targeted gene knock-out. In this chapter, we describe ATMT protocols and vectors for fungal gene manipulation using two plant pathogenic fungi, Magnaporthe grisea and Fusarium oxysporum, as target organisms.

Global Expression Profiling of Transcription Factor Genes Provides New Insights into Pathogenicity and Stress Responses in the Rice Blast Fungus

Because most efforts to understand the molecular mechanisms underpinning fungal pathogenicity have focused on studying the function and role of individual genes, relatively little is known about how transcriptional machineries globally regulate and coordinate the expression of a large group of genes involved in pathogenesis. Using quantitative real-time PCR, we analyzed the expression patterns of 206 transcription factor (TF) genes in the rice blast fungus Magnaporthe oryzae under 32 conditions, including multiple infection-related developmental stages and various abiotic stresses. The resulting data, which are publicly available via an online platform, provided new insights into how these TFs are regulated and potentially work together to control cellular responses to a diverse array of stimuli. High degrees of differential TF expression were observed under the conditions tested. More than 50% of the 206 TF genes were up-regulated during conidiation and/or in conidia. Mutations in ten conidiation-specific TF genes caused defects in conidiation. Expression patterns in planta were similar to those under oxidative stress conditions. Mutants of in planta inducible genes not only exhibited sensitive to oxidative stress but also failed to infect rice. These experimental validations clearly demonstrated the value of TF expression patterns in predicting the function of individual TF genes. The regulatory network of TF genes revealed by this study provides a solid foundation for elucidating how M. oryzae regulates its pathogenesis, development, and stress responses.

Cellular and Molecular Analyses of Biotrophic Invasion in Rice Blast Disease

To cause rice blast disease, Magnaporthe oryzae sequentially invades living plant cells using intracellular invasive hyphae (IH) that grow from cell to cell. However, detailed cellular and molecular mechanisms underlying biotrophic invasion are poorly understood. We used live-cell microscopy and fluorescent molecular probes to visualize biotrophic invasion of rice sheath epidermal cells, and demonstrated that IH are sealed in a plant-derived Extra-Invasive-Hyphal Membrane (EIHM) as they grow in first-invaded rice cells and then spread into neighboring cells. The fungus appears to manipulate plasmodesmata for its cell-to-cell movement, based in part on searching behavior of IH before crossing the plant cell wall and on extreme constriction of IH as they cross. Studies of transformed fungal strains that secrete avirulence effector:green fluorescent protein fusions in rice sheath cells led to discovery of a novel pathogen-induced structure, the Biotrophic Interfacial Complex (BIC), that appears to play a role in effector secretion. Optimization of the sheath assay for molecular analysis allowed us to analyze infected rice tissue in which 20% of the RNA came from IH growing in first-invaded cells. This allowed identification of novel candidate effectors by microarray analysis. Next comes understanding dual roles for blast effectors in promoting disease or, in the case of avirulence effectors recognized by rice resistance gene products, in blocking disease.