Role In Asexual Development Research Articles

Histone H2B lysine 122 and lysine 130, as the putative targets of Penicillium oxalicum LaeA, play important roles in asexual development, expression of secondary metabolite gene clusters, and extracellular glycoside hydrolase synthesis.

Core histones in the nucleosome are subject to a wide variety of posttranslational modifications (PTMs), such as methylation, phosphorylation, ubiquitylation, and acetylation, all of which are crucial in shaping the structure of the chromatin and the expression of the target genes. A putative histone methyltransferase LaeA/Lae1, which is conserved in numerous filamentous fungi, functions as a global regulator of fungal growth, virulence, secondary metabolite formation, and the production of extracellular glycoside hydrolases (GHs). LaeA's direct histone targets, however, were not yet recognized. Previous research has shown that LaeA interacts with core histone H2B. Using S-adenosyl-L-methionine (SAM) as a methyl group donor and recombinant human histone H2B as the substrate, it was found that Penicillium oxalicum LaeA can transfer the methyl groups to the C-terminal lysine (K) 108 and K116 residues in vitro. The H2BK108 and H2BK116 sites on recombinant histone correspond to P. oxalicum H2BK122 and H2BK130, respectively. H2BK122A and H2BK130A, two mutants with histone H2B K122 or K130 mutation to alanine (A), were constructed in P. oxalicum. The mutants H2BK122A and H2BK130A demonstrated altered asexual development and decreased extracellular GH production, consistent with the findings of the laeA gene deletion strain (ΔlaeA). The transcriptome data showed that when compared to wild-type (WT) of P. oxalicum, 38 of the 47 differentially expressed (fold change ≥ 2, FDR ≤ 0.05) genes that encode extracellular GHs showed the same expression pattern in the three mutants ΔlaeA, H2BK122A, and H2BK130A. The four secondary metabolic gene clusters that considerably decreased expression in ΔlaeAalso significantly decreased in H2BK122A or H2BK130A. The chromatin of promotor regions of the key cellulolytic genes cel7A/cbh1 and cel7B/eg1 compacted in the ΔlaeA, H2BK122A, and H2BK130A mutants, according to the results of chromatin accessibility real-time PCR (CHART-PCR). The chromatin accessibility index dropped. The histone binding pocket of the LaeA-methyltransf_23 domain is compatible with particular histone H2B peptides, providing appropriate electrostatic and steric compatibility to stabilize these peptides, according to molecular docking. The findings of the study demonstrate that H2BK122 and H2BK130, which are histone targets of P. oxalicum LaeA in vitro, are crucial for fungal conidiation, the expression of gene clusters encoding secondary metabolites, and the production of extracellular GHs.

Roles of two new transcription factors in the synergistic effect of combined cinnamaldehyde and nonanal against Aspergillus flavus

Aspergillus flavus, an opportunistic pathogen, is renowned for colonizing agricultural crops, leading to aflatoxin contamination. In this study, the inhibitory potency of combined cinnamaldehyde and nonanal (SCAN) against A. flavus on stored grains was evaluated, and the mechanism of action at the molecular level was explored. SCAN exhibited synergistic action against A. flavus on wheat, resulting in a 99.17% decrease in spore production. This was clearly a greater decrease than if the individual compounds were used separately. RNA-sequencing and gene knock-out analyses were performed to clarify the synergistic mechanism. Transcriptome data revealed that two new transcription factors (TFs), TF-1 (AFLA_049760) and TF-2 (AFLA_048110), were down-regulated when exposed to 0.03 μL/mL and 0.45 μL/mL SCAN. The TF-1 and TF-2 deletion mutants presented slower growth rates compared with wild-type A. flavus. In addition, conidial production was decreased by down-regulating the conidiation genes brlA and abaA. Stress assays and confocal laser scanning microscopic examinations revealed that more reactive oxygen species accumulated in ΔTF-1 mutants compared with wild-type A. flavus. These two TFs positively regulated the production of aflatoxin B1 biosynthesis and down-regulated the expression of genes, including aflM, aflQ, aflR and aflS, in the aflatoxin gene cluster. More importantly, our results indicated that the mutants were impaired in their ability to infect crop seeds. Thus, the two new TFs, which play key roles in asexual development, aflatoxin B1 biosynthesis and fungal virulence in A. flavus, may be the critical regulators in the fungus after exposure to SCAN.

Achog1 is required for the asexual sporulation, stress responses and pigmentation of Aspergillus cristatus.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea; hypotonic conditions only induce its sexual development to produce ascospores, while hypertonic conditions only induce its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the role of Achog1, which is homologous to hog1 from Saccharomyces cerevisiae, in sporulation, different kinds of stress responses and pigment production was investigated. Deletion mutants of Achog1 were obtained by homologous recombination. Phenotypic observations showed that the time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in the deletion mutants of Achog1 in hypertonic media, indicating that Achog1 plays a positive role in asexual development. Stress sensitivity tests showed that ΔAchog1 strains were sensitive to hyperosmolarity, and the order of the sensitivity of ΔAchog1 to different osmotic regulators was 3 M sucrose >3 M NaCl >3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. pH sensitivity tests indicated that Achog1 inhibited the growth of A. cristatus under alkaline stress. Additionally, pigmentation was decreased in the Achog1 deletion mutants compared with the WT. All the above developmental defects were reversed by the reintroduction of the Achog1 gene in ΔAchog1. Pull-down and LC-MS/MS analysis showed that the expression levels of proteins interacting with Achog1 were significantly different under low and high osmotic stress, and proteins related to conidial development were present only in the cultures treated with hyperosmotic stress. Transcription profiling data showed that Achog1 suppressed the expression of several genes related to asexual development, osmotic and oxidative stress resistance. On the basis of gene knockout, pull-down mass spectrometry and RNA-seq analyses, a regulatory pathway for Achog1 was roughly identified in A. cristatus.

BHLH transcription factor EcdR controls conidia production, pigmentation and virulence in Aspergillus fumigatus

Invasive Aspergillus fumigatus infection is a disease with high morbidity and mortality rates. Abnormalities in sporulation and pigmentation can significantly alter the pathogenicity of A. fumigatus, thus the mechanisms of conidiation and pigment biosynthesis have gained increasing attention. In Aspergillus oryzae, a novel predicted bHLH protein-encoding gene, ecdR, plays a role in asexual development, and its ortholog has also been characterized in A. nidulans. Herein, we determined its role in A. fumigatus by testing whether ecdR deletion affects asexual development, melanin synthesis, and regulation of virulence in this fungus. Our study shows that EcdR controls conidia and melanin production in A. fumigatus. In addition, we found that virulence in the ΔecdR strain was significantly reduced in the infection model of immunodeficiency mice.

The role of Acpbs2 in the asexual sporulation, stress responseand carbon metabolism of Aspergillus cristatus.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea, hypotonic conditions only induced its sexual development to produce ascospores, while hypertonic conditions only induced its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the roles of Acpbs2, which is homologous to pbs2 from Saccharomyces cerevisiae, in sporulation, stress responses, the color of colonies, and carbon metabolism were explored in A. cristatus. Deletion mutants of Acpbs2 were obtained by homologous recombination. The time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in hypertonic media in ΔAcpbs2 by phenotypic observations, indicating that Acpbs2 plays a positive role in asexual development. Stress sensitivity tests showed that the order of the sensitivity of ΔAcpbs2 to different osmotic regulators was 3 M NaCl > 3 M sucrose > 3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. The growth of the Acpbs2 deletion mutant was inhibited under alkaline-pH stress, indicating that Acpbs2 is involved in high pH stress tolerance. Additionally, compared with the wild type, the colony color of the Acpbs2 deletion mutant became lighter. All the above developmental defects were reversed by the reintroduction of the Acpbs2 gene in ΔAcpbs2. Transcriptome data showed that Acpbs2 regulated the expression of several genes related to conidial development, osmotic stress, oxidative stress, and carbon metabolism. More importantly, the interaction between Acpbs2 and its downstream gene Achog1 was verified by yeast two-hybrid assays. We speculated that this interaction might regulate the osmotic stress response, the oxidative stress response, and asexual sporulation in A. cristatus, which will be one of the focuses of our future research.

Roles and Cellular Localization of GBP2 and NAB2 During the Blood Stage of Malaria Parasites.

The quality control and export of mRNA by RNA-binding proteins are necessary for the survival of malaria parasites, which have complex life cycles. Nuclear poly(A) binding protein 2 (NAB2), THO complex subunit 4 (THO4), nucleolar protein 3 (NPL3), G-strand binding protein 2 (GBP2) and serine/arginine-rich splicing factor 1 (SR1) are involved in nuclear mRNA export in malaria parasites. However, their roles in asexual and sexual development, and in cellular localization, are not fully understood. In this study using the rodent malaria parasite, Plasmodium berghei, we found that NAB2 and SR1, but not THO4, NPL3 or GBP2, played essential roles in the asexual development of malaria parasites. By contrast, GBP2 but not NPL3 was involved in male and female gametocyte production. THO4 was involved in female gametocyte production, but had a lower impact than GBP2. In this study, we focused on GBP2 and NAB2, which play important roles in the sexual and asexual development of malaria parasites, respectively, and examined their cellular localization. GBP2 localized to both the nucleus and cytoplasm of malaria parasites. Using immunoprecipitation coupled to mass spectrometry (IP-MS), GBP2 interacted with the proteins ALBA4, DOZI, and CITH, which play roles in translational repression. IP-MS also revealed that phosphorylated adapter RNA export protein (PHAX) domain-containing protein, an adaptor protein for exportin-1, also interacted with GBP2, implying that mRNA export occurs via the PHAX domain-containing protein pathway in malaria parasites. Live-cell fluorescence imaging revealed that NAB2 localized at the nuclear periphery. Moreover, IP-MS indicated that NAB2 interacted with transportin. RNA immunoprecipitation coupled to RNA sequencing revealed that NAB2 bound directly to 143 mRNAs, including those encoding 40S and 60S ribosomal proteins. Our findings imply that malaria parasites use an evolutionarily ancient mechanism conserved throughout eukaryotic evolution.

Requirement of LaeA, VeA, and VelB on Asexual Development, Ochratoxin A Biosynthesis, and Fungal Virulence in Aspergillus ochraceus.

Aspergillus ochraceus is reported to be the major contributor of ochratoxin A (OTA), classified as one of the possible human carcinogen (group 2B) by the International Agency for Research on Cancer. The heterotrimeric velvet complex proteins, LaeA/VeA/VelB, have been most studied in fungi to clarify the relation between light-dependent morphology and secondary metabolism. To explore possible genetic targets to control OTA contamination, we have identified laeA, veA, and velB in A. ochraceus. The loss of laeA, veA, and velB yielded mutants with differences in vegetative growth and conidial production. Especially, ΔlaeA almost lost the ability to generate conidiaphore under dark condition. The deletion of laeA, veA, and velB drastically reduced the production of OTA. The wild-type A. ochraceus produced about 1 and 7 μg/cm2 OTA under light and dark conditions on media, whereas the three gene deletion mutants produced less than 20 ng/cm2 OTA, which was correlated with a down regulation of OTA biosynthetic genes. Pathogenicity studies of ΔlaeA, ΔveA, and ΔvelB showed their reduction in disease severity in pears. Furthermore, 66.1% of the backbone genes in secondary metabolite gene cluster were significantly regulated, among which 81.6% were downregulated. Taking together, these results revealed that velvet complex proteins played crucial roles in asexual development, secondary metabolism, and fungal virulence in A. ochraceus.

FvSTR1, a striatin orthologue in Fusarium virguliforme, is required for asexual development and virulence.

The soil-borne fungus Fusarium virguliforme causes sudden death syndrome (SDS), one of the most devastating diseases of soybean in North and South America. Despite the importance of SDS, a clear understanding of the fungal pathogenicity factors that affect the development of this disease is still lacking. We have identified FvSTR1, a F. virguliforme gene, which encodes a protein similar to a family of striatin proteins previously reported to regulate signalling pathways, cell differentiation, conidiation, sexual development, and virulence in filamentous fungi. Striatins are multi-domain proteins that serve as scaffolding units in the striatin-interacting phosphatase and kinase (STRIPAK) complex in fungi and animals. To address the function of a striatin homologue in F. virguliforme, FvSTR1 was disrupted and functionally characterized using a gene knock out strategy. The resulting Fvstr1 mutants were largely impaired in conidiation and pigmentation, and displayed defective conidia and conidiophore morphology compared to the wild-type and ectopic transformants. Greenhouse virulence assays revealed that the disruption of FvSTR1 resulted in complete loss of virulence in F. virguliforme. Microtome studies using fluorescence microscopy showed that the Fvstr1 mutants were defective in their ability to colonize the vascular system. The Fvstr1 mutants also showed a reduced transcript level of genes involved in asexual reproduction and in the production of secondary metabolites. These results suggest that FvSTR1 has a critical role in asexual development and virulence in F. virguliforme.

Fungal Histidine Phosphotransferase Plays a Crucial Role in Photomorphogenesis and Pathogenesis in Magnaporthe oryzae.

Two-component signal transduction (TCST) pathways play crucial roles in many cellular functions such as stress responses, biofilm formation, and sporulation. The histidine phosphotransferase (HPt), which is an intermediate phosphotransfer protein in a two-component system, transfers a phosphate group to a phosphorylatable aspartate residue in the target protein(s), and up-regulates stress-activated MAP kinase cascades. Most fungal genomes carry a single copy of the gene coding for HPt, which are potential antifungal targets. However, unlike the histidine kinases (HK) or the downstream response regulators (RR) in two-component system, the HPts have not been well-studied in phytopathogenic fungi. In this study, we investigated the role of HPt in the model rice-blast fungal pathogen Magnaporthe oryzae. We found that in M. oryzae an additional isoform of the HPT gene YPD1 was expressed specifically in response to light. Further, the expression of light-regulated genes such as those encoding envoy and blue-light-harvesting protein, and PAS domain containing HKs was significantly reduced upon down-regulation of YPD1 in M. oryzae. Importantly, down-regulation of YPD1 led to a significant decrease in the ability to penetrate the host cuticle and in light-dependent conidiation in M. oryzae. Thus, our results indicate that Ypd1 plays an important role in asexual development and host invasion, and suggest that YPD1 isoforms likely have distinct roles to play in the rice-blast pathogen M. oryzae.

Role of the Talaromyces marneffei (Penicillium marneffei) sakA gene in nitrosative stress response, conidiation and red pigment production.

Stress-activated MAPK pathways are systems used to regulate the stress adaptation of most fungi. It has been shown that in Talaromyces marneffei (Penicillium marneffei), a pathogenic dimorphic fungus, the sakA gene is involved, not only in tolerance against oxidative and heat stresses, but also in playing a role in asexual development, yeast cell generation in vitro and survival inside macrophage cell lines. In this study, the role of the T. marneffei sakA gene on the nitrosative stress response and the regulation of gene expression were investigated. The susceptibility of the sakA mutant to NaNO2 was investigated using drop dilution assay and the expression of genes of interest were determined by RT-PCR, comparing them to the wild-type and complemented strains. The results demonstrated that the T. marneffei sakA gene played a role in the stress response to NaNO2, the expression of genes functioning in conidial development (brlA, abaA and wetA) and red pigment biosynthesis (pks3, rp1, rp2 and rp3). These findings suggest that T. marneffei sakA is broadly involved in a wide variety of cell activities, including stress response, cell morphogenesis, asexual development and pigmentation.

BcMtg2 is required for multiple stress tolerance, vegetative development and virulence in Botrytis cinerea.

In Saccharomyces cerevisiae, the Mtg2 gene encodes the Obg protein, which has an important function in assembling ribosomal subunits. However, little is known about the role of the Obg GTPase in filamentous fungi. In this study, we identified an Mtg2 ortholog, BcMtg2, in B. cinerea. The BcMtg2 deletion mutant showed a defect in spore production, conidial germination and sclerotial formation. Additionally, the mutant increased sensitivity to various environmental stresses. The BcMtg2 mutant exhibited dramatically decreased virulence on host plant tissues. BcMtg2 mutant showed increased sensitivity to osmotic and oxidative stresses, and to Congo red (cell wall stress agent). In the yeast complement assay, growth defects of yeast BY4741ΔMTG2 mutant were partly restored by genetic complementation of BcMtg2 under these environmental stresses. Additionally, compared with the parental strain and complement strain, the BcMtg2 deletion mutant displayed a minor glycerol response to osmosis stress. These defective phenotypes were recovered in the complement strain ΔBcMtg2C, which was created by adding the wild-type BcMtg2 gene to the ΔBcMtg2 mutant. The results of this study indicate that BcMtg2 has a necessary role in asexual development, environmental stress response and pathogenicity in B. cinerea.

Characterization of sakA gene from pathogenic dimorphic fungus Penicillium marneffei

Eukaryotes utilize stress activated protein kinase (SAPK) pathways to adapt to environmental stress, including heat, osmotic, oxidative or nutrient stresses. Penicillium marneffei (Talaromyces marneffei), the dimorphic pathogenic fungus that can cause disseminated mycosis in HIV-infected patients, has to encounter various types of stresses both outside and inside host cells. However, the strategies used by this fungus in response to these stresses are still unclear. In this report, the stress-activated kinase (sakA) gene of P. marneffei was characterized and the roles of this gene on various stress conditions were studied. The sakA gene deletion mutant was constructed using the split marker method. The phenotypes and sensitivities to varieties of stresses, including osmotic, oxidative, heat and cell wall stresses of the deletion mutant were compared with the wild type and the sakA complemented strains. Results demonstrated that the P. marneffei sakA gene encoded a putative protein containing TXY phosphorylation lip found in the stress high osmolarity glycerol 1 (Hog1)/Spc1/p38 MAPK family, and that this gene was involved not only in tolerance against oxidative and heat stresses, but also played a role in asexual development, chitin deposition, yeast cell generation in vitro and survival inside mouse and human macrophages.

The N-terminus region of the putative C2H2 transcription factor Ada1 harbors a species-specific activation motif that regulates asexual reproduction in Fusarium verticillioides

Fusarium verticillioides is an important plant pathogenic fungus causing maize ear and stalk rots. In addition, the fungus is directly associated with fumonisin contamination of food and feeds. Here, we report the functional characterization of Ada1, a putative Cys2-His2 zinc finger transcription factor with a high level of similarity to Aspergillus nidulans FlbC, which is required for the activation of the key regulator of conidiation brlA. ADA1 is predicted to encode a protein with two DNA binding motifs at the C terminus and a putative activator domain at the N terminus region. Deletion of the flbC gene in A. nidulans results in “fluffy” cotton-like colonies, with a defect in transition from vegetative growth to asexual development. In this study we show that Ada1 plays a key role in asexual development in F. verticillioides. Conidia production was significantly reduced in the knockout mutant (Δada1), in which aberrant conidia and conidiophores were also observed. We identified genes that are predicted to be downstream of ADA1, based on A. nidulans conidiation signaling pathway. Among them, the deletion of stuA homologue, FvSTUA, resulted in near absence of conidia production. To further investigate the functional conservation of this transcription factor, we complemented the Δada1 strain with A. nidulans flbC, F. verticillioides ADA1, and chimeric constructs. A. nidulans flbC failed to restore conidia production similar to the wild-type level. However, the Ada1N-terminal domain, which contains a putative activator, fused to A. nidulans FlbC C-terminal motif successfully complemented the Δada1 mutant. Taken together, Ada1 is an important transcriptional regulator of asexual development in F. verticillioides and that the N-terminus domain is critical for proper function of this transcription factor.

A serine/threonine-protein phosphatase PP2A catalytic subunit is essential for asexual development and plant infection in Magnaporthe oryzae

Protein phosphatase 2A is a subgroup of widely conserved serine/threonine phosphatases and plays diverse roles in transcription, translation, differentiation, cell cycle, and signal transduction in many organisms. However, its roles in biotrophic and hemi-biotrophic phytopathogenic fungi remain to be investigated. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that was defective in vegetative hyphal growth. In the mutant, the T-DNA fragment was found to be inserted in the promoter region of a putative serine/threonine protein phosphatase 2A catalytic subunit (PP2Ac) gene MoPPG1. Deletion of MoPPG1 leads to severe defects in vegetative hyphal growth and conidiation. Conidia of the ∆Moppg1 null mutants were misshaped, and most of them were two-celled. The deletion mutants of MoPPG1 did not penetrate into host plant cells and failed to cause any disease lesions on rice leaves. Interestingly, significant reduction was found in the ∆Moppg1 null mutants in expression levels of several Rho GTPase family genes including MgCDC42, MgRHO3, and MgRAC1, which were important for pathogenesis of M. oryzae. Taken together, our results indicated that PP2Ac plays vital roles in asexual development and plant infection by regulating Rho GTPases in the rice blast fungus and perhaps other plant pathogenic fungi.

NsdC and NsdD Affect Aspergillus flavus Morphogenesis and Aflatoxin Production

The transcription factors NsdC and NsdD are required for sexual development in Aspergillus nidulans. We now show these proteins also play a role in asexual development in the agriculturally important aflatoxin (AF)-producing fungus Aspergillus flavus. We found that both NsdC and NsdD are required for production of asexual sclerotia, normal aflatoxin biosynthesis, and conidiophore development. Conidiophores in nsdC and nsdD deletion mutants had shortened stipes and altered conidial heads compared to those of wild-type A. flavus. Our results suggest that NsdC and NsdD regulate transcription of genes required for early processes in conidiophore development preceding conidium formation. As the cultures aged, the ΔnsdC and ΔnsdD mutants produced a dark pigment that was not observed in the wild type. Gene expression data showed that although AflR is expressed at normal levels, a number of aflatoxin biosynthesis genes are expressed at reduced levels in both nsd mutants. Expression of aflD, aflM, and aflP was greatly reduced in nsdC mutants, and neither aflatoxin nor the proteins for these genes could be detected. Our results support previous studies showing that there is a strong association between conidiophore and sclerotium development and aflatoxin production in A. flavus.

Early lineage specification of long-lived germline precursors in the colonial ascidian Botryllus schlosseri.

In many taxa, germline precursors segregate from somatic lineages during embryonic development and are irreversibly committed to gametogenesis. However, in animals that can propagate asexually, germline precursors can originate in adults. Botryllus schlosseri is a colonial ascidian that grows by asexual reproduction, and on a weekly basis regenerates all somatic and germline tissues. Embryonic development in solitary ascidians is the classic example of determinative specification, and we are interested in both the origins and the persistence of stem cells responsible for asexual development in colonial ascidians. In this study, we characterized vasa as a putative marker of germline precursors. We found that maternally deposited vasa mRNA segregates early in development to a posterior lineage of cells, suggesting that germline formation is determinative in colonial ascidians. In adults, vasa expression was observed in the gonads, as well as in a population of mobile cells scattered throughout the open circulatory system, consistent with previous transplantation/reconstitution results. vasa expression was dynamic during asexual development in both fertile and infertile adults, and was also enriched in a population of stem cells. Germline precursors in juveniles could contribute to gamete formation immediately upon transplantation into fertile adults, thus vasa expression is correlated with the potential for gamete formation, which suggests that it is a marker for embryonically specified, long-lived germline progenitors. Transient vasa knockdown did not have obvious effects on germline or somatic development in adult colonies, although it did result in a profound heterochrony, suggesting that vasa might play a homeostatic role in asexual development.

Penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects.

Penicillium marneffei infection is an important emerging public health problem, especially among patients infected with human immunodeficiency virus in the areas of endemicity in southeast Asia, India, and China. Within these regions, P. marneffei infection is regarded as an AIDS-defining illness, and the severity of the disease depends on the immunological status of the infected individual. Early diagnosis by serologic and molecular assay-based methods have been developed and are proving to be important in diagnosing infection. The occurrence of natural reservoirs and the molecular epidemiology of P. marneffei have been studied; however, the natural history and mode of transmission of the organism remain unclear. Soil exposure, especially during the rainy season, has been suggested to be a critical risk factor. Using a highly discriminatory molecular technique, multilocus microsatellite typing, to characterize this fungus, several isolates from bamboo rats and humans were shown to share identical multilocus genotypes. These data suggest either that transmission of P. marneffei may occur from rodents to humans or that rodents and humans are coinfected from common environmental sources. These putative natural cycles of P. marneffei infection need further investigation. Studies on the fungal genetics of P. marneffei have been focused on the characterization of genetic determinants that may play important roles in asexual development, mycelial-to-yeast phase transition, and the expression of antigenic determinants. Molecular studies have identified several genes involved in germination, hyphal development, conidiogenesis, and yeast cell polarity. A number of functionally important genes, such as the malate synthase- and catalase-peroxidase protein-encoding genes, have been identified as being upregulated in the yeast phase. Future investigations pertaining to the roles of these genes in host-fungus interactions may provide the key knowledge to understanding the pathogenicity of P. marneffei.