Infection Hyphae Research Articles

Septation of Infectious Hyphae Is Critical for Appressoria Formation and Virulence in the Smut Fungus Ustilago Maydis

Differentiation of hyphae into specialized infection structures, known as appressoria, is a common feature of plant pathogenic fungi that penetrate the plant cuticle. Appressorium formation in U. maydis is triggered by environmental signals but the molecular mechanism of this hyphal differentiation is largely unknown. Infectious hyphae grow on the leaf surface by inserting regularly spaced retraction septa at the distal end of the tip cell leaving empty sections of collapsed hyphae behind. Here we show that formation of retraction septa is critical for appressorium formation and virulence in U. maydis. We demonstrate that the diaphanous-related formin Drf1 is necessary for actomyosin ring formation during septation of infectious hyphae. Drf1 acts as an effector of a Cdc42 GTPase signaling module, which also consists of the Cdc42-specific guanine nucleotide exchange factor Don1 and the Ste20-like kinase Don3. Deletion of drf1, don1 or don3 abolished formation of retraction septa resulting in reduced virulence. Appressorium formation in these mutants was not completely blocked but infection structures were found only at the tip of short filaments indicating that retraction septa are necessary for appressorium formation in extended infectious hyphae. In addition, appressoria of drf1 mutants penetrated the plant tissue less frequently.

An unusual method of development and sporulation of Colletotrichum gloeosporioides on castor leaf – an SEM account

Development and sporogenesis of Colletotrichum gloeosporioides on castor leaf differed from that on other known host plants. C. gloeosporioides had three kinds of hyphae on castor leaf: primary infection hyphae (PIH), runner hyphae (RH) and secondary infection hyphae (SIH). The PIH originated from conidia, grew on leaf surface and entered the leaf by direct penetration of the cuticle without forming appressoria. The RH were sub-cuticular hyphae, the track of which was traceable by the bulgings on the leaf surface, and the SIH were the hyphae that emerged to leaf surface from RH through the cuticle or stomata. Conidia were initiated as small protrusions along the lengths of RH and SIH that got differentiated into distinct conidia, each born on a short stumpy conidiophore without forming any congregation. The protrusions from RH emerged to the leaf surface by piercing the cuticle, and they developed into distinct conidia on the leaf surface. The conidia developed from RH and SIH were identical in size and shape. Even though conidia were occasionally found emerged through stomata, that appeared to be random than a preferred route for the discharge of conidia. The penetration and sporogenesis of C. gloeosporioides on castor leaf differed from that reported on mulberry leaf.

Auxin Production by the Rice Blast Fungus and its Localization in Host Tissue

AbstractThe rice blast fungus Magnaporthe oryzae requires living plant cells in its early stages of infection and invasion of host tissue. In this study, we showed the possibility that an exogenous fungal auxin could be implicated in hemibiotrophic interactions. Chemical analyses showed that this fungus produces a small amount of the auxin indole‐3‐acetic acid during growth in culture. Immunohistological analyses showed that antigens of the anti‐indole‐3‐acetic acid monoclonal antibody were localized in the fungal infection hyphae present in the rice tissue. Rice transformants containing an auxin‐inducible reporter construct demonstrated an auxin response around the apex of the infection hyphae after fungal invasion of living tissue. These results suggest that the rice blast fungus produces a small amount of auxin during its biotrophic phase and the invaded rice tissue responds to the exogenous fungal auxin. This auxin may be involved in the biotrophic phase of the infection hyphae.

Laser Capture Microdissection of Uredinia Formed by Melampsora larici-populina Revealed a Transcriptional Switch Between Biotrophy and Sporulation

The foliar rust caused by the basidiomycete Melampsora larici-populina is the main disease affecting poplar plantations in Europe. The biotrophic status of rust fungi is a major limitation to study gene expression of cell or tissue types during host infection. At the uredinial stage, infected poplar leaves contain distinct rust tissues such as haustoria, infection hyphae, and uredinia with sporogenous hyphae and newly formed asexual urediniospores. Laser capture microdissection (LCM) was used to isolate three areas corresponding to uredinia and subjacent zones in the host mesophyll for expression analysis with M. larici-populina whole-genome exon oligoarrays. Optimization of tissue preparation prior to LCM allowed isolation of RNA of good integrity for genome-wide expression profiling. Our results indicate that the poplar rust uredinial stage is marked by distinct genetic programs related to biotrophy in the host palisade mesophyll and to sporulation in the uredinium. A strong induction of transcripts encoding small secreted proteins, likely containing rust effectors, is observed in the mesophyll, suggesting a late maintenance of suppression of host defense in the tissue containing haustoria and infection hyphae. On the other hand, cell cycle and cell defense rescue transcripts are strongly accumulated in the sporulation area. This combined LCM-transcriptomic approach brings new insights on the molecular mechanisms underlying urediniospore formation in rust fungi.

Perception of the chitin oligosaccharides contributes to disease resistance to blast fungus Magnaporthe oryzae in rice

Chitin is a component of fungal cell walls, and its fragments act as elicitors in many plants. The plasma membrane glycoprotein CEBiP, which possesses LysM domains, is a receptor for the chitin elicitor (CE) in rice. Here, we report that the perception of CE by CEBiP contributes to disease resistance against the rice blast fungus, Magnaporthe oryzae, and that enhanced responses to CE by engineering CEBiP increase disease tolerance. Knockdown of CEBiP expression allowed increased spread of the infection hyphae. To enhance defense responses to CE, we constructed chimeric genes composed of CEBiP and Xa21, which mediate resistance to rice bacterial leaf blight. The expression of either CRXa1 or CRXa3, each of which contains the whole extracellular portion of CEBiP, the whole intracellular domain of XA21, and the transmembrane domain from either CEBiP or XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after treatment with CE. Rice plants expressing the chimeric receptor exhibited necrotic lesions in response to CE and became more resistant to M.oryzae. Deletion of the first LysM domain in CRXA1 abolished these cellular responses. These results suggest that CEs are produced and recognized through the LysM domain of CEBiP during the interaction between rice and M.oryzae and imply that engineering pattern recognition receptors represents a new strategy for crop protection against fungal diseases.

Cellular and transcriptional responses of wheat during compatible and incompatible race‐specific interactions with Puccinia striiformis f. sp. tritici

The initial stages of Puccinia striiformis f. sp. tritici (the causal agent of yellow rust in wheat) infection triggered a hypersensitive cell death (HCD) response in both compatible and Yr1-mediated incompatible interactions, although the response was earlier and more extensive in the incompatible interaction. Later stages of fungal development were only associated with an HCD response in the incompatible interaction, the HCD response being effectively suppressed in the compatible interaction. Cell autofluorescence was seen in mesophyll cells in direct contact with fungal infection hyphae (primary HCD) and in adjacent mesophyll cells (secondary HCD), indicating the activation of cell-to-cell signalling. Microarray analysis identified a number of defence-related transcripts implicated in Yr1-mediated resistance, including classical pathogenesis-related (PR) transcripts and genes involved in plant cell defence responses, such as the oxidative burst and cell wall fortification. A quantitative reverse transcriptase-polymerase chain reaction time course analysis identified a number of defence-related genes, including PR2, PR4, PR9, PR10 and WIR1 transcripts, associated with the latter stages of Yr1-mediated resistance. A meta-analysis comparison of the Yr1-regulated transcriptome with the resistance transcriptomes of the race-specific resistance gene Yr5 and the race-nonspecific adult plant resistance gene Yr39 indicated limited transcript commonality. Common transcripts were largely confined to classic PR and defence-related genes.

The Basic Leucine Zipper Transcription Factor Moatf1 Mediates Oxidative Stress Responses and Is Necessary for Full Virulence of the Rice Blast Fungus Magnaporthe oryzae

Magnaporthe oryzae is the causal agent of rice blast disease, leading to enormous losses of rice production. Here, we characterized a basic leucine zipper (bZIP) transcription factor, Moatf1, in M. oryzae, a homolog of Schizosaccharomyces pombe ATF/CREB that regulates the oxidative stress response. Moatf1 deletion caused retarded vegetative growth of mycelia, and the Moatf1 mutant exhibited higher sensitivity to hydrogen peroxide (H(2)O(2)) than did the wild-type strain. The mutant showed severely reduced activity of extracellular enzymes and transcription level of laccases and peroxidases and exhibited significantly reduced virulence on rice cultivar CO-39. On rice leaf sheath, most of the infectious hyphae of the mutant became swollen and displayed restricted growth in primary infected cells. Defense response was strongly activated in plants infected by the mutant. Diamino benzidine staining revealed an accumulation of H(2)O(2) around Moatf1 mutant appressoria and rice cells with Moatf1 hyphae that was absent in the wild type. Inhibition of the plant NADPH oxidase by diphenyleneiodonium prevented host-derived H(2)O(2) accumulation and restored infectious hyphal growth of the mutant in rice cells. Thus, we conclude that Moatf1 is necessary for full virulence of M. oryzae by regulating the transcription of laccases and peroxidases to impair reactive oxygen species-mediated plant defense.

U romyces appendiculatus Infection in BTH-Treated Bean Plants: Ultrastructural Details of a Lost Fight

The mechanisms of BTH [benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester]-induced resistance against bean rust caused by Uromyces appendiculatus have been explored in Phaseolus vulgaris by light and transmission electron microscopy, following the infection progression in plants challenged 7 days after treatment. While BTH did not affect uredospore germination and fungal penetration in the substomatal cavity, a first impairment to the colonization appeared evident about 48-96 h after inoculation, with alterations of infection hypha structure and reduction in mycelium expansion. No differences were found in this phase regarding the formation and ultrastructure of haustoria in untreated and BTH-treated plants, except for the deposition of electron-opaque material in the extrahaustorial matrix of the latter. A second and decisive impairment in fungal progression was observed at 7-10 days after inoculation when host cell penetrated, or in close contact with the fungal hyphae, were impregnated by phenolic compounds. The same was observed in fungal walls, particularly around haustoria, thus hampering the biotrophic habitus of the fungus and further mycelium spreading. This, in turn, prevented the evasion of fungal reproductive structures, the uredinia, and the appearance of visible symptoms. No particular ultrastructural alterations were observed in most of the penetrated cells, even at late stages of infection, indicating that BTH treatment does not induce host cells to respond with a hypersensitive reaction (HR). A parallel time course of the expression of phenylalanine ammonia lyase (PAL) gene, the key enzyme for the synthesis of phenylpropanoidic phytoalexins and many other phenolics, has shown that PAL mRNA is strongly and persistently transcripted in BTH-treated plants since the 6th h after treatment, though no apparent ultrastructural alterations were detectable up to some days after pathogen challenging. This indicates that BTH, at the employed concentration of 0.3 mM, directly activates the plant's own defences, thus accounting for the observed full protection against bean rust.

Dynamics of arbuscular mycorrhizal fungi and glomalin in the rhizosphere of Artemisia ordosica Krasch. in Mu Us sandland, China

To understand the ecological significance of arbuscular mycorrhizal (AM) associations in semi-arid and arid lands, the temporal and spatial dynamics of AM fungi and glomalin were surveyed in Mu Us sandland, northwest China. Soil samples in the rhizosphere of Artemisia ordosica Krasch. were collected in May, July and October 2007, respectively. Arbuscular, hyphal and total root infection and spore density of AM fungi peaked in summer. The mean contents of total Bradford-reactive soil proteins (T-BRSPs, TG) and easily extractable Bradford-reactive soil proteins (EE-BRSPs, EEG) reached maximal values in spring. Spore density and two BRSPs fractions were the highest in the 0–10 cm soil layer, but the ratios of two BRSPs fractions to soil organic carbon (SOC) were the highest in the 30–50 cm soil layer. Hyphal infection was negatively correlated with soil enzymatic activity (soil urease and acid phosphatase) ( P < 0.05). Arbuscular infection was negatively correlated with soil acid phosphatase ( P < 0.01). Spore density was positively correlated with edaphic factors (soil available N, Olsen P, and SOC) and soil enzymatic activity (soil acid and alkaline phosphatase) ( P < 0.01). Two BRSPs fractions were positively correlated with edaphic factors (soil available N and SOC) and soil enzymatic activity (soil urease, acid and alkaline phosphatase) ( P < 0.01). TG was positively correlated with soil Olsen P ( P < 0.05). We concluded that the dynamics of AM fungi and glomalin have highly temporal and depth patterns, and influenced by nutrient availability and enzymatic activity in Mu Us sandland, and suggest that glomalin are useful indicators for evaluating soil quality and function of desert ecosystem on the basis of its relationship to AM fungal community, soil nutrient dynamics and carbon cycle.

An in vitro method for the analysis of infection‐related morphogenesis in Fusarium graminearum

Fusarium graminearum is a significant pathogen of many cereal crops. With its genetic tractability, ease of culture, genome sequence availability and economic significance, F. graminearum has become the subject of intensive molecular research. Although molecular tools have been developed to enhance research into virulence determinants of F. graminearum, simple assays for infection-related development are lacking. As such, the objective of this study was to develop an in vitro protocol for the analysis of infection-related morphogenesis in F. graminearum. We demonstrate that two morphologically distinct hyphal structures are produced by F. graminearum during the invasion of detached wheat glumes: subcuticular hyphae and bulbous infection hyphae. Specialized wheat epidermal cells (papillae) appear to act as sites of invasion by F. graminearum on the adaxial side of detached wheat glumes. In addition, the development of bulbous infection hyphae is dependent on the pathogenicity mitogen-activated protein kinase Gpmk1, further supporting the infection-related nature of these structures. This relatively simple assay will contribute to the tractability of the F. graminearum system and help to uncover molecular requirements for infection-related development.

A Kelch Repeat Protein, Cokel1p, Associates with Microtubules and Is Involved in Appressorium Development in Colletotrichum orbiculare

Kelch repeat proteins are conserved in diverse organisms and some are known to mediate fundamental cellular functions. We isolated the gene CoKEL1, encoding a novel kelch repeat protein, from Colletotrichum orbiculare. Analysis of a cokel1 mutant indicated that CoKEL1 is involved in proper appressorium development and cell wall synthesis. Appressoria produced by cokel1 disruption mutants showed irregular shape and impairment of turgor generation and the mutant appressoria rarely penetrated to form infection hyphae in host epidermal cells. Accordingly, cokel1 mutants had reduced pathogenicity on host leaves compared with the wild type. Furthermore, the cokel1 mutant was more sensitive to cell-wall-degrading enzymes and showed altered labeling with the cell wall stain Calcofluor white. Cokel1p was localized on cortical and spindle microtubules in vegetative hyphae. These results suggest that Cokel1p is a microtubule-associated protein involved in infection-related morphogenesis and pathogenicity. This is the first report that a kelch repeat protein is required for the pathogenicity of a fungal plant pathogen.

Scanning Electron Microscopy of Invasion Process by Fusarium Moniliforme on Soybean Seed

Fusarium moniliforme J. Sheld was observed as important seed-borne pathogen of soybean (Glycine max) having a great potentiality to cause seed-borne infection after establishing a rapid pathogenesis. The infection process was studied with scanning electron microscopy (SEM). Infection hyphae grew directly from the side or near the end of the conidium and entered the seed coat through cuticular cell juncture with or without forming appressorium. After invasion of the fungus, rapid degradation of cell wall occurred followed by intercellular and intracellular development of the fungus. Finally, tissues of the seed lost their integrity and identity and seemed as rotten mass covering with dense mycelium.

Dynamics of cell wall components of Magnaporthe grisea during infectious structure development

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea. alpha-1,3-glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and beta-1,3-glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were alpha-1,3-glucan and chitosan, but after enzymatic digestion of alpha-1,3-glucan, beta-1,3-glucan and chitin became detectable. Immunoelectron microscopic analysis showed alpha-1,3-glucan and beta-1,3-glucan intermixed in the cell wall of infectious hyphae; however, alpha-1,3-glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of alpha-1,3-glucan. Accumulation of alpha-1,3-glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16-hexadecanediol. Taken together, alpha-1,3-glucan spatially and functionally masks beta-1,3-glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea.

The Supernatant of a Conidial Suspension of Magnaporthe oryzae Contains a Factor that Promotes the Infection of Rice Plants

AbstractMany factors produced by the pathogen Magnaporthe oryzae enhance its ability to infect rice. We found a novel infection‐promoting activity in the supernatant of a conidia suspension (SCS) of M. oryzae. The addition of SCS promoted the invasion of excised rice leaf sheaths by infectious hyphae. The activity was heat‐stable and was found in SCSs from five virulent and three avirulent isolates of M. oryzae on the rice cv. Nipponbare (Pia). The effect was exclusively detected in compatible interactions. The infection of rice plants by non‐rice blast fungi was not enhanced by SCS. These results suggest that SCS includes a heat‐stable factor(s) that promotes M. oryzae infection during compatible interactions.

Diversity of defence mechanisms in plant–oomycete interactions: a case study of Lactuca spp. and Bremia lactucae

Plant pathogenic oomycetes, including biotrophic downy mildews and hemibiotrophs/necrotrophs such as Phytophthora and Pythium, cause enormous economic losses on cultivated crops. Lettuce breeders and growers face the threat of Bremia lactucae, the causal agent of lettuce downy mildew. This pathogen damages leaf tissues and lettuce heads and is also frequent on wild Asteraceae plants. The interactions of Lactuca spp. with B. lactucae (abbr. lettuce–Bremia) display extreme variability, due to a long co-evolutionary history. For this reason, during the last 30 years, the lettuce–Bremia pathosystem has been used as a model for many studies at the population, individual, organ, tissue, cellular, physiological and molecular levels, as well as on genetic variability and the genetics of host–parasite interactions. The first part of this review summarizes recent data on host–parasite specificity, host variability, resistance mechanisms and genetics of lettuce–Bremia interactions. The second part focuses on the development infection structures. Phenotypic expression of infection, behaviour of B. lactucae on leaf surfaces, the process of penetration, development of primary infection structures, hyphae and haustoria are discussed in relation to different resistance mechanisms. In the third part, the components of host resistance and the variability of defence responses are analysed. The role of reactive oxygen species (ROS), antioxidant enzymes, nitric oxide (NO), phenolic compounds, reorganization of cytoskeleton, electrolyte leakage, membrane damage, cell wall disruption, hypersensitive reaction and plant energetics are discussed in relation to defence responses. In general, the extreme variability of interactions between lettuce and Bremia, and their phenotypic expression, results from diversity of the genetic background. Different mechanisms of resistance are conditioned by an orchestra of defence responses at the tissue, cell, and molecular levels. The various events responsible for defence involve a complex interaction of the processes and reactions mentioned above. This review also provides an overview on the timing of pathogen development, host pathological anatomy, cytology and physiology of lettuce–Bremia associations. The significance of these factors on the expression of different resistance mechanisms (non-host and host resistance, race-specific and race non-specific resistance, field resistance) is discussed.

Infection process of Colletotrichum dematium on mulberry leaves: An unusual method of sporulation

The pre-penetration and infection process of Colletotrichum dematium on mulberry leaf was investigated by scanning electron microscope. Conidia produced on germination appressoria directly or at the end of short germ tubes. Appressoria were formed mostly over cuticle, but sometimes over stomata also. At 72 h post-inoculation, an extensive network of sub-cuticular runner hyphae (RH) was produced. The RH were traceable by the cuticular bulgings on leaf surface. The RH emerged to leaf surface through ruptured cuticle to form secondary infection hyphae (SIH). The SIH re-entered the leaf tissue by sending penetration branches through stomata. Conidia were formed singly on short conidiophores from the RH and SIH, at short intervals. The conidia developed on RH were exposed to leaf surface through ruptured cuticle. Some times conidia were released through stomata also. The RH and SIH had thick knots from which hyphal branches and conidia were developed. Definite acervuli were not developed.

MADS-Box Transcription Factor Mig1 Is Required for Infectious Growth in Magnaporthe grisea

Magnaporthe grisea is a model fungus for studying fungus-plant interactions. Two mitogen-activated protein (MAP) kinase genes, PMK1 and MPS1, have been implicated in regulating plant infection processes in M. grisea. However, transcription factors activated by these MAP kinases are not well studied. In this study we functionally characterized the MIG1 gene that encodes a MADS-box transcription factor homologous to Saccharomyces cerevisiae Rlm1. In yeast two-hybrid assays, MIG1 interacts with MPS1, suggesting that MIG1 may function downstream from the MPS1 pathway. The mig1 deletion mutant had a normal growth rate and formed melanized appressoria, but it was nonpathogenic and failed to infect rice leaves through wounds. Appressoria formed by the mig1 mutant developed penetration pegs and primary infectious hyphae, but further differentiation of the secondary infectious hyphae inside live plant cells was blocked. However, the mig1 mutant formed infectious hypha-like structures in heat-killed plant cells or cellophane membranes. In transformants expressing the MIG1-GFP fusion, green fluorescent protein (GFP) signals were not detectable in vegetative hyphae and conidiophores. Mig1-GFP was localized to nuclei in conidia, appressoria, and infectious hyphae. Deletion of the MADS box had no effect on the expression and localization of the MIG1-GFP fusion but eliminated its ability to complement the mig1 mutant. These results suggest that MIG1 may be required for overcoming plant defense responses and the differentiation of secondary infectious hyphae in live plant cells. The MADS-box domain is essential for the function of MIG1 but dispensable for its nuclear localization, which may be associated with the activation of MIG1 by MPS1 during conidiation and plant infection.

Polar growth in the infectious hyphae of the phytopathogen ustilago maydis depends on a virulence-specific cyclin.

The maize smut fungus Ustilago maydis switches from yeast to hyphal growth to infect maize (Zea mays) plants. This switching is promoted by mating of compatible cells and seems to be required for plant penetration. Although many genes distinctively expressed during this dimorphic switch have been identified and shown to be essential for the infection process, none seems to be explicitly required for polar growth control. Here, we report the characterization of pcl12, encoding a cyclin that interacts specifically with Cdk5, an essential cyclin-dependent kinase with regulatory roles in morphogenesis in U. maydis. Pcl12 fulfills the requirements to be a virulence-specific regulator of polar growth in U. maydis. First, pcl12 expression is induced during the pathogenic development. Secondly, Pcl12 is sufficient to induce hyperpolarized growth in U. maydis cells, as haploid cells overexpressing pcl12 in axenic conditions produce filaments that were morphologically indistinguishable from those produced during the infection process. Finally, cells defective in pcl12 showed impaired polar growth during the formation of the b-dependent filament, the induction of the conjugation tubes, or the formation of a promycelium in spore germination. However, in spite of this pivotal role during morphogenesis, pcl12 mutants were virulent. We discuss the implications of these results for the role of polar growth during the infection process.

Mir1 is Highly Upregulated and Localized to Nuclei During Infectious Hyphal Growth in the Rice Blast Fungus

Rice blast, caused by Magnaporthe grisea, is a devastating disease of rice throughout the world. Many recent molecular studies have focused on the early infection stages, but our knowledge about molecular events at the infectious hyphae stage is limited. In this study, 750 hygromycin-resistant transformants were isolated by transforming M. grisea Guyll with a promoterless enhanced green fluorescent protein (EGFP) construct. In one of the transformants, L1320, EGFP signals were observed in the nuclei of infectious hyphae. The transforming vector was inserted in a predicted gene named MIR1 and resulted in a Mir1 1-107-EGFP fusion. Mir1 is a low-complexity protein with no known protein domain and has no homolog in GenBank or other sequenced fungal genomes. Quantitative real-time reverse-transcriptase polymerase chain reaction analysis and expression assays of MIR1-EGFP fusion constructs indicated that the expression of MIR1 was highly induced during plant infection. Deletion analyses identified a 458-bp region that was sufficient for the MIR1 promoter activity. Further characterization revealed that a 96-bp sequence was essential for the enhanced in planta expression. MIR1 is an M. grisea-specific gene that is highly conserved among the field isolates belonging to the M. grisea species complex. The mir1 mutants had no obvious defects in appressorial penetration and rice infection. When overexpressed with the RP27 promoter, nuclear localization of the Mir1-EGFP fusion was observed in conidia and vegetative hyphae. These data suggest that the expression but not the nuclear localization of MIR1 is specific to infectious hyphae and that reporter genes based on MIR1 may be suitable for monitoring infectious growth in M. grisea.

The RNA-binding protein Rrm4 is essential for polarity inUstilago maydisand shuttles along microtubules

Formation of polar-growing hyphae is essential for infection by the plant pathogen Ustilago maydis. Here we observe that loss of RNA-recognition motif protein Rrm4 caused formation of abnormal hyphae. The insertion of septa at the distal pole was abolished and a significantly increased number of hyphae grew bipolarly. UV-crosslinking experiments revealed that Rrm4 bound RNA via its N-terminal RRMs and that its RNA-binding activity was substantially increased during filamentation. Rrm4 assembled into particles that shuttled bidirectionally along microtubules to both poles. Recruitment of Rrm4 into particles increased during filamentation, and mutations in the peptide-binding pocket of its PABC domain caused abnormal particle formation as well as polarity defects. Shuttling was mediated by active transport because loss of conventional kinesin, which interferes with the balance of microtubule-dependent motors, caused accumulation of particles at the poles resulting in disturbed polarity. Thus, constant transport of the RNA-binding protein towards the poles is needed to orchestrate hyphal growth. Since a mutation of the N-terminal RRM that leads to reduced RNA binding in vivo also affected polarity, Rrm4 might regulate polarity of the infectious hyphae by transporting RNA from the nucleus to cell poles.