Many fungal pathogens develop specialized infection structures such as appressoria to penetrate plant cells. However, it is not clear whether special structures are formed after cell wall penetration before invading host cytoplasm membrane in hemibiotrophic pathogens. Here, we showed that a penetration ring consisting of Ppe1 secreted proteins is formed after appressorium-mediated cell wall penetration and remained at the base of penetration site after invading plant cytoplasm membrane in the rice blast fungus Magnaporthe oryzae . The same persistent Ppe1 ring is formed after the penetration of neighboring cells by transpressoria. PPE1 is specifically expressed during plant infection and the Δ ppe1 mutant is defective in penetration and invasive growth. Blockage of penetration peg formation impedes the development of the Ppe1 ring. Close examinations showed that the penetration ring is formed at the collar of penetration pegs between plant cell wall and cytoplasm membrane and it is persistent as a fixed ring even after invasive hyphae invaded neighboring cells. Furthermore, Ppe1 is a member of an expanded family of secreted proteins that are unique to fungal pathogens using extreme appressorium turgor for plant penetration. Other members of the Ppe1 family also localize to the penetration ring for anchoring on cytoplasm membrane during plant infection. Taken together, a penetration ring consisting of a family of secreted proteins is formed between plant cell wall and cytoplasm membrane, which may function as a novel physical structure at the interface between the tip of penetration pegs and plant cytoplasm membrane before the differentiation of invasive hyphae.

Abstract Plant-pathogen interactions are governed by the ability of phytopathogens to utilize specialized infection structures and effectors for successful invasion. The rice blast disease, caused by the fungus Magnaporthe oryzae, poses a significant threat to global food production. In this study, we investigate the role of a novel apoplastic effector, peg periphery-localized effector (Ppe), in M. oryzae invasion. We demonstrate that Ppe is specifically expressed during plant infection and exhibits dynamic spatiotemporal localizations. It is preferentially localized to a ring structure at the collar of penetration peg formed by appressoria or transpressoria although occasionally observed in vesicle-like structures within the biotrophic interfacial complex (BIC). Through the unconventional secretion pathway involving the exocyst complex and t-SNAREs, Ppe is delivered to the rice apoplastic interface. Genetic analysis reveals that while the Δppe mutant does not exhibit developmental differences compared to the wild type, it displays reduced pathogenicity due to defects in rice penetration and expansion. Furthermore, Ppe suppresses the expression of plant defense-related genes and attenuates callose deposition at plasmodesmata, thereby facilitating fungal invasion. Our findings highlight the key role of Ppe as a novel effector in M. oryzae invasion and emphasize the significance of the peg as an infection structure and effector deployment site for overcoming host defense responses.

Seventy host-adapted gene ( HAG) effector family members from Pyricularia species are found in P. oryzae and three closely related species (isolates LS and 18-2 from an unknown Pyricularia sp., P. grisea, and P. pennisetigena) that share at least eight orthologous HAG family members with P. oryzae. The genome sequence of a more distantly related species, P. penniseti, lacks HAG genes, suggesting a time frame for the origin of the gene family in the genus . In P. oryzae, HAG4 is uniquely found in the genetic lineage that contains populations adapted to Setaria and Oryza hosts. We find a nearly identical HAG4 allele in a P. grisea isolate, suggesting transfer of HAG4 from P. grisea to P. oryzae. HAG4 encodes a suppressor of plant cell death. Yeast two-hybrid screens with several HAG genes independently identify common interacting clones from a rice complementary DNA library, suggesting conservation of protein surface motifs between HAG homologs with as little as 40% protein sequence identity. HAG family orthologs have diverged rapidly and HAG15 orthologs display unusually high rates of sequence divergence compared with adjacent genes suggesting gene-specific accelerated divergence. The sequence diversity of the HAG homologs in Pyricularia species provides a resource for examining mechanisms of gene family evolution and the relationship to structural and functional evolution of HAG effector family activity. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Plant pathogen effectors play important roles in parasitism, including countering plant immunity. However, investigations of the emergence and diversification of fungal effectors across host-adapted populations has been limited. We previously identified a gene encoding a suppressor of plant cell death in Pyricularia oryzae (syn. Magnaporthe oryzae). Here, we report the gene is one of a 21-member gene family and we characterize sequence diversity in different populations. Within the rice pathogen population, nucleotide diversity is low, however; the majority of gene family members display presence-absence polymorphism or other null alleles. Gene family allelic diversity is greater between host-adapted populations and, thus, we named them host-adapted genes (HAGs). Multiple copies of HAGs were found in some genome assemblies and sequence divergence between the alleles in two cases suggested they were the result of repeat-induced point mutagenesis. Transfer of family members between populations and novel HAG haplotypes resulting from apparent recombination were observed. HAG family transcripts were induced in planta and a subset of HAGs are dependent on a key regulator of pathogenesis, PMK1. We also found differential intron splicing for some HAGs that would prevent ex planta protein expression. For some genes, spliced transcript was expressed in antiphase with an overlapping antisense transcript. Characterization of HAG expression patterns and allelic diversity reveal novel mechanisms for HAG regulation and mechanisms generating sequence diversity and novel allele combinations. This evidence of strong in planta–specific expression and selection operating on the HAG family is suggestive of a role in parasitism. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Secondary metabolites (SMs) are crucial for fungi and vary in function from beneficial antibiotics to pathogenicity factors. To generate diversified SMs that enable different functions, SM-coding regions rapidly evolve in fungal genomes. However, the driving force and genetic mechanism of fungal SM diversification in the context of host-pathogen interactions remain largely unknown. Previously, we grouped field populations of the rice blast fungus Magnaporthe oryzae (syn: Pyricularia oryzae) into three major globally distributed clades based on population genomic analyses. Here, we characterize a recent duplication of an avirulent gene-containing SM cluster, ACE1, in a clonal M. oryzae population (Clade 2). We demonstrate that the ACE1 cluster is specifically duplicated in Clade 2, a dominant clade in indica rice-growing areas. With long-read sequencing, we obtained chromosome-level genome sequences of four Clade 2 isolates, which displayed differences in genomic organization of the ACE1 duplication process. Comparative genomic analyses suggested that the original ACE1 cluster experienced frequent rearrangement in Clade 2 isolates and revealed that the new ACE1 cluster is located in a newly formed and transposable element-rich region. Taken together, these results highlight the frequent mutation and expansion of an avirulent gene-containing SM cluster through transposable element-mediated whole-cluster duplication in the context of host-pathogen interactions.

The genome of rice blast fungus (Magnaporthe oryzae) encodes 15 glycoside hydrolase 18 family chitinases. In this study, we characterized the function of an M. oryzae extracellular chitinase, MoChi1, and its interaction with a host protein, OsMBL1, a jacalin-related Mannose-Binding Lectin (MBL) in rice (Oryza sativa). Deletion of MoChi1 resulted in reduced aerial hyphal formation and reduced virulence in rice by activating the expression of defense-responsive genes. We confirmed MoChi1 interaction with rice OsMBL1 in vitro and in vivo. OsMBL1 was induced by pathogen-associated molecular patterns and M. oryzae infection. Overexpression of OsMBL1 led to activation of rice defense-responsive genes and a chitin-induced reactive oxygen species burst, thereby enhancing resistance to M. oryzae. Knockdown of OsMBL1 enhances susceptibility of rice plants to M. oryzae. Furthermore, MoChi1 suppressed chitin-induced reactive oxygen species in rice cells and competed with OsMBL1 for chitin binding. Taken together, our study reveals a mechanism in which MoChi1 targets a host lectin to suppress rice immunity.

Magnaporthe oryzae (Mo) is a model pathogen causing rice blast resulting in yield and economic losses world-wide. CK2 is a constitutively active, serine/threonine kinase in eukaryotes, having a wide array of known substrates, and involved in many cellular processes. We investigated the localization and role of MoCK2 during growth and infection. BLAST search for MoCK2 components and targeted deletion of subunits was combined with protein-GFP fusions to investigate localization. We found one CKa and two CKb subunits of the CK2 holoenzyme. Deletion of the catalytic subunit CKa was not possible and might indicate that such deletions are lethal. The CKb subunits could be deleted but they were both necessary for normal growth and pathogenicity. Localization studies showed that the CK2 holoenzyme needed to be intact for normal localization at septal pores and at appressorium penetration pores. Nuclear localization of CKa was however not dependent on the intact CK2 holoenzyme. In appressoria, CK2 formed a large ring perpendicular to the penetration pore and the ring formation was dependent on the presence of all CK2 subunits. The effects on growth and pathogenicity of deletion of the b subunits combined with the localization indicate that CK2 can have important regulatory functions not only in the nucleus/nucleolus but also at fungal specific structures such as septa and appressorial pores.

We examined the genomes of 100 isolates of Magnaporthe oryzae ( Pyricularia oryzae ), the causal agent of rice blast disease. We grouped current field populations of M . oryzae into three major globally distributed groups. A genetically diverse group, clade 1, which may represent a group of closely related lineages, contains isolates of both mating types. Two well-separated clades, clades 2 and 3, appear to have arisen as clonal lineages distinct from the genetically diverse clade. Examination of genes involved in mating pathways identified clade-specific diversification of several genes with orthologs involved in mating behavior in other fungi. All isolates within each clonal lineage are of the same mating type. Clade 2 is distinguished by a unique deletion allele of a gene encoding a small cysteine-rich protein that we determined to be a virulence factor. Clade 3 isolates have a small deletion within the MFA2 pheromone precursor gene, and this allele is shared with an unusual group of isolates we placed within clade 1 that contain AVR1-CO39 alleles. These markers could be used for rapid screening of isolates and suggest specific events in evolution that shaped these populations. Our findings are consistent with the view that M. oryzae populations in Asia generate diversity through recombination and may have served as the source of the clades 2 and 3 isolates that comprise a large fraction of the global population.

Current approaches for obtaining homokaryons of asexually growing N. crassa rely on serial passages of macroconidia, which are usually multinuclear (Davis and de Serres 1970 Methods Enzymol. 17A:79143). This method is time consuming and labor intensive. We have devised a simple method for purifying viable uninucleate microconidia from conidiating strains of N. crassa grown on Westergaard and Mitchell synthetic crossing medium (SC) supplemented with iodoacetate (IAA). Rossier, Oulevey and Turian (1973 Arch. Mikrobiol. 91:345-353) showed that standing liquid cultures containing 1x SC and 1 mM IAA produced microconidia. We have modified their method to reliably obtain microconidia from 150 mm slants of solid agar medium (0.1 x SC/0.5% sucrose/2% agar/1 mM IAA). We have purified these microconidia free of macroconidia and mycelia using Millipore Durapore Millex 5 μm filters. We are using this technique for the one step purification of homokaryons following DNA-mediated transformation. These methods may be generally useful for studies with heterokaryons. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol37/iss1/7 A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia Ebbole, D. and M.S. Sachs Department of Biological Sciences, Stanford University, Stanford, CA 94305 Current approaches for obtaining homokaryons of asexually growing N. crassa rely on serial passages of macroconidia, which are usually multinuclear (Davis and de Serres 1970 Methods Enzymol. 17A:79143). This method is time consuming and labor intensive. We have devised a simple method for purifying viable uninucleate microconidia from conidiating strains of N. crassa grown on Westergaard and Mitchell synthetic crossing medium (SC) supplemented with iodoacetate (IAA). Rossier, Oulevey and Turian (1973 Arch. Mikrobiol. 91:345-353) showed that standing liquid cultures containing 1x SC and 1 mM IAA produced microconidia. We have modified their method to reliably obtain microconidia from 150 mm slants of solid agar medium (0.1 x SC/0.5% sucrose/2% agar/1 mM IAA). We have purified these microconidia free of macroconidia and mycelia using Millipore Durapore Millex 5 μm filters. We are using this technique for the one step purification of homokaryons following DNA-mediated transformation. These methods may be generally useful for studies with heterokaryons. EXPERIMENTAL Sterile 16 x 150 mm glass culture tubes containing 6 ml of a sterile, molten solu-tion consisting of 2% agar/0.5% sucrose/0.1 x SC and plugged with pre-autoclaved foam plugs, were adjusted to 50-60°C in a water bath. Then 60 μl of 0.1 M sodium IAA (Sigma catalog # I2512, freshly prepared, filter-sterilized in water) was mixed with the contents of each tube (final concentration 1.0 mM). Tubes were slanted at room temperature until solidified, and stored refrigerated for up to one week. Slants were inoculated using an agar plug of mycelia (obtained by coring a colony with a sterile pasteur pipet) or using a drop of macroconidia suspended in water. Cultures were incubated at 25°C with a 12 hour light/dark cycle for 7-10 days. The production of microconidia was checked by lightly scraping an area of the sparse surface growth (away from tufts of macroconidia) with a sterile, wet inoculating loop. The loopful of culture was transferred to 20 μl of water and examined microscopically. Microconidia were harvested from cultures by adding 2.5 ml of sterile water to tubes followed by rigorous vortex mixing for 30-60 sec (more microconidia were obtainable by repeating this harvesting step). The conidial suspensions were passed through 5 μm Millex Durapore filter units (Millipore catalog number SLSV025LS) using sterile conditions. Typically 0.1-1% of the microconidia were recovered. The yield as determined by counting with a hemacytometer varied from 10(3)-10(6) microconidia per slant. Filtrates with low numbers of microconidia were concentrated by pelleting the microconidia in a clinical centrifuge (Beckman RT6000, 2000 x g, 5 min). Most of each supernatant was removed by aspiration and the pellets resuspended in the remaining liquid. Microconidia were germinated at 34°C after spreading on freshly prepared Vogel's/sorbose agar plates. Microconidial viability varied from 1-20%, as determined by comparison of the number Published by New Prairie Press, 2017 of colony forming centers/ml after seven days growth to the number of microconidia/ml as determined by direct counting. We typically plated 2000 microconidia/plate to obtain homokaryotic cultures, and picked colonies after 2-3 days. Vegetative homokaryotic stocks were obtained by transferring individual colonies to slants of Vogel's sucrose medium. RESULTS AND DISCUSSION We analyzed purified microconidia microscopically using the fluorescent dye Hoechst 33258 to stain nuclei (Springer and Yanofsky 1989 Genes. Dev. 3:559-571). Purified microconidia were almost all uninucleate, although microconidia containing two nuclei and enucleated microconidia were also seen. In contrast, macroconidia from Vogel's minimal slants mostly contained 2-5 nuclei, and macroconidia from the slants of 0.1 x SC/0.5% sucrose/2% agar/1 mM IAA mostly contained 1-3 nuclei. Genetic analyses of microconidia were consistent with the nuclear counting data. Microconidia were obtained from the forced heterokaryon cyh-1 ad-3B am1 + al-1 lys-4 a grown on minimal medium. Equal numbers of microconidia (2000/plate) gave the following numbers of colonies when assayed on the four media shown below: Medium Plates Colonies Colonies/Plate ade + lys 5 81 16.2 ade 7 62 8.8 lys 10 48 4.8 minimal 10 1 0.1 These results demonstrate that this method effectively resolved this heterokaryon into its individual ade-requiring and lys-requiring components. The single colony obtained on the minimal plate was subsequently shown to be heterokaryotic in origin, based on analysis of it microconidia. We have been routinely using microconidia to obtain homokaryons of strains transformed with exogenously applied DNA. Homokaryotic His+ transformants of Hisstrains were readily obtainable in a single step by selecting for the growth of His+ microconidia on unsupplemented media. Drug resistant transformants could be identified by subsequent screening of cultures grown nonselectively, or by plating microconidia directly on drug-supplemented plates. Microconidia derived from hygromycin B resistant strains transformed with several hph expression constructs readily formed colonies when germinated on plates containing hygromycin B. When plated on benomyl-containing medium (1 μg/ml), microconidia derived from a benomyl-resistant homokaryon germinated poorly (25% control value) and the colonies obtained grew slowly. Seventeen control colonies obtained by germination of the microconidia on drugfree plates were subsequently tested for benomyl resistance: all were benomyl resistant, as expected. We find that IAA is essential for increased production of microconidia although the mechanism by which it acts is not known (Rossier et al. 1973). We have not examined whether IAA can be autoclaved in the media. When comparing SC concentrations we found 0.1 x SC to give more reproducible results than 0.5 x or 1.0 x SC. We have not tested production of microconidia on slants containing any additional supplements. Growth at 34°C appears to reduce the number of http://newprairiepress.org/fgr/vol37/iss1/7 DOI: 10.4148/1941-4765.1472 microconidia produced. We have not tried lower growth temperatures, or tested whether the light/dark cycle is necessary for microconidial development. Nylon 66 (Schleicher and Schuell) with 5 μm pore size can also be used for this procedure but we have more limited experience with it. We don't know precisely the features responsible for the differential filtration of conidia. The poor yield of microconidia suggests that the separation by these membranes may involve properties in addition to pore size. In general, the filtered microconidia germinate over a period of 1-3 days. However, it is prudent to wait for 3 days to be sure that when a colony is picked it is not contaminated with a late germinating microconidium. Microconidia germinate equally well at 34°C or 25°C. CONCLUSION We believe from our preliminary observations that this technique can be of immediate use to Neurospora workers. We have routinely used it successfully to isolate homokaryons from primary transformants and have verified that we have obtained homokaryons by Southern analyses. Using our procedure we can pick a colony from a transformation plate and in 10 days obtain a homokaryon using only one 150 mm slant, one filtration step, one plate, and very little of the investigator's time. ACKNOWLEDGEMENTS We thank Charles Yanofsky and David Perkins for advice and use of equipment. We wish to acknowledge the advice and technical assistance of Brian White. D. Ebbole and M. Sachs contributed equally to this work. Published by New Prairie Press, 2017

Carbon catabolite repression (CCR) is a common regulatory mechanism used by microorganisms to prioritize use of a preferred carbon source (usually glucose). The CreC WD40-repeat protein is a major component of the CCR pathway in Aspergillus nidulans. To clarify the function of the CreC ortholog from Magnaporthe oryzae in regulating gene expression important for pathogenesis, MoCreC was identified and genetically characterized. The vegetative growth rate of the MoCreC deletion mutant on various carbon sources was reduced. The MoCreC mutant produced fewer conidia and with about 60% of conidia having septation defects. Appressorium formation was impaired in the MoCreC mutant. Although some appressoria of the mutant could penetrate the leaf surface successfully, the efficiency of penetration and invasive growth of infection hyphae was reduced, resulting in attenuated virulence toward host plants. The CCR was defective as the mutant was more sensitive to allyl alcohol in the presence of glucose, and 2-deoxyglucose was unable to fully repress utilization of secondary carbon sources. qRT-PCR results indicated that the genes encoding cell wall degradation enzymes, such as β-glucosidase, feruloyl esterase and exoglucanase, were upregulated in MoCreC mutant. Taken together, we conclude that MoCreC is a major regulator of CCR and plays significant roles in regulating growth, conidiation, and pathogenicity of M. oryzae.

Rice blast disease, caused by Magnaporthe oryzae, threatens global food security. The rice blast pathosystem is a longstanding model system for understanding plant-microbe interactions. In order to elucidate the coevolution of the host and pathogen, and provide the appropriate methods for preventing or controlling rice blast disease, researchers have focused on the evolution of virulence factors and resistance genes. Thus far, more than 30 rice blast resistance (R) genes and 12 avirulence (Avr) genes have been cloned. This review summarizes the cloned rice blast R genes, cloned Avr genes of M. oryzae and the interaction between them. This discussion also considers some of the major unanswered questions concerning this pathosystem and the opportunities for future investigations.

One major threat to global food security that requires immediate attention, is the increasing incidence of host shift and host expansion in growing number of pathogenic fungi and emergence of new pathogens. The threat is more alarming because, yield quality and quantity improvement efforts are encouraging the cultivation of uniform plants with low genetic diversity that are increasingly susceptible to emerging pathogens. However, the influence of host genome differentiation on pathogen genome differentiation and its contribution to emergence and adaptability is still obscure. Here, we compared genome sequence of 6 isolates of Magnaporthe species obtained from three different host plants. We demonstrated the evolutionary relationship between Magnaporthe species and the influence of host differentiation on pathogens. Phylogenetic analysis showed that evolution of pathogen directly corresponds with host divergence, suggesting that host-pathogen interaction has led to co-evolution. Furthermore, we identified an asymmetric selection pressure on Magnaporthe species. Oryza sativa-infecting isolates showed higher directional selection from host and subsequently tends to lower the genetic diversity in its genome. We concluded that, frequent gene loss or gain, new transposon acquisition and sequence divergence are host adaptability mechanisms for Magnaporthe species, and this coevolution processes is greatly driven by directional selection from host plants.

Rab GTPases represent the largest subfamily of Ras-related small GTPases and regulate membrane trafficking. Vesicular transport is a general mechanism that governs intracellular membrane trafficking along the endocytic and exocytic pathways in all eukaryotic cells. Fusarium graminearum is a filamentous fungus and causes the devastating and economically important head blight of wheat and related species. The mechanism of vesicular transport is not well understood, and little is known about Rab GTPases in F. graminearum. In this study, we systematically characterized all eleven FgRabs by live cell imaging and genetic analysis. We find that FgRab51 and FgRab52 are important for the endocytosis, FgRab7 localizes to the vacuolar membrane and regulates the fusion of vacuoles and autophagosomes, and FgRab8 and FgRab11 are important for polarized growth and/or exocytosis. Furthermore, both endocytic and exocytic FgRabs are required for vegetative growth, conidiogenesis, sexual reproduction, as well as pathogenesis and deoxynivalenol metabolism in F. graminearum. Thus, we conclude that Rab GTPases are essential for membrane trafficking-dependent growth and pathogenicity in F. graminearum.

The retromer mediates protein trafficking through recycling cargo from endosomes to the trans-Golgi network in eukaryotes. However, the role of such trafficking events during pathogen-host interaction remains unclear. Here, we report that the cargo-recognition complex (MoVps35, MoVps26 and MoVps29) of the retromer is essential for appressorium-mediated host penetration by Magnaporthe oryzae, the causal pathogen of the blast disease in rice. Loss of retromer function blocked glycogen distribution and turnover of lipid bodies, delayed nuclear degeneration and reduced turgor during appressorial development. Cytological observation revealed dynamic MoVps35-GFP foci co-localized with autophagy-related protein RFP-MoAtg8 at the periphery of autolysosomes. Furthermore, RFP-MoAtg8 interacted with MoVps35-GFP in vivo, RFP-MoAtg8 was mislocalized to the vacuole and failed to recycle from the autolysosome in the absence of the retromer function, leading to impaired biogenesis of autophagosomes. We therefore conclude that retromer is essential for autophagy-dependent plant infection by the rice blast fungus.

•Localization of exocyst components differs at the point of polarized growth.•The Spitzenkörper has evolved during speciation to optimize hyphal growth.•The exocyst is used in different pathways to deliver effectors to plant cells.•Fungi have evolved different secretory routes and/or regulatory mechanisms.

Aspergillus nidulans StuA and Neurospora crassa ASM-1 are orthologous APSES (ASM-1, PHD1, SOK2, Efg1, StuA) transcription factors conserved across a diverse group of fungi. StuA and ASM-1 have roles in asexual (conidiation) and sexual (ascospore formation) development in both organisms. To address the hypothesis that the last common ancestor of these diverse fungi regulated conidiation with similar genes, asm-1 was introduced into the stuA1 mutant of A. nidulans. Expression of asm-1 complemented defective conidiophore morphology and restored conidia production to wild type levels in stuA1. Expression of asm-1 in the stuA1 strain did not rescue the defect in sexual development. When the conidiation regulator AbaA was tagged at its C-terminus with GFP in A. nidulans, it localized to nuclei in phialides. When expressed in the stuA1 mutant, AbaA::GFP localized to nuclei in conidiophores but no longer was confined to phialides, suggesting that expression of AbaA in specific cell types of the conidiophore was conditioned by StuA. Our data suggest that the function in conidiation of StuA and ASM-1 is conserved and support the view that, despite the great morphological and ontogenic diversity of their condiphores, the last common ancestor of A. nidulans and N. crassa produced an ortholog of StuA that was involved in conidiophore development.

G-protein-coupled receptors (GPCRs) are an important receptor gene family and play important roles in sensing sugars in eukaryotic organisms, including fungi. Recent studies on the interactions between Cryptococcus and plants, a major environmental niche, reveal that myo-inositol, produced and secreted by plants, is sensed by the fungus and promotes fungal sexual reproduction, providing a potential explanation for how this organism completes its life cycle in nature. Several fusel alcohols, such as 1-butanol and isoamyl alcohol, stimulate filamentous growth of haploid cells. A recent study showed that aromatic alcohols (such as tryptophol and phenylethanol) secreted by yeast cells function as quorum-sensing molecules and stimulate filamentous growth through a Flo11-dependent mechanism. Amino acids are important nutrients for fungi and are detected by specialized sensor systems, which include the general amino acid permease Gap1, the Ssy1-Ptr3-Ssy5 (SPS) system, GPCRs, and the target of rapamycin (TOR). In Neurospora crassa, three transport systems have been described based on the analysis of the kinetics of amino acid uptake and the patterns of competitive inhibition between amino acids. The Gap1 homolog in Neurospora crassa is encoded by the PMG locus, which can transport all L-amino acids except proline. Besides this system, two other transport systems have also been identified. One is encoded by the MTR gene and transports neutral and aromatic amino acids. The other is encoded by the PMB gene and transports basic amino acids, such as arginine and lysine.

Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression, massively parallel signature sequencing, and sequencing by synthesis to examine transcriptome profiles of infected rice leaves. A total of 6,413 in planta-expressed fungal genes, including 851 genes encoding predicted effector proteins, were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. Four of them induced cell death in Nicotiana benthamiana. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in planta-expressed cell death-inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.