Induce Appressorium Formation Research Articles

AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata.

Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6h) and a 1.46-fold upregulation during the infection hyphae formation period (8h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.

Molecular Characterization of Phospholipase C in Infection Structure Differentiation Induced by Pear Fruit Surface Signals, Stress Responses, Secondary Metabolism, and Virulence of Alternaria alternata.

Fungal pathogens use plant surface physiochemical signals to trigger specific developmental processes. To assess the role of phospholipase C (PLC) in mediating plant stimuli sensing of Alternaria alternata, the function of three PLC genes was characterized by constructing ΔAaPLC mutants. Here we showed that fruit wax-coated surfaces significantly induced appressorium formation in A. alternata and mutants. Germination of ΔAaPLC mutants did not differ from the wild type. Deletion of AaPLC1 led to the decrease of appressorium formation and infected hyphae, but the degree of reduction varies between the different types of waxes, with the strongest response to pear wax. Appressorium formation and infected hyphae of the ΔAaPLC1 mutant on dewaxed onion epidermis mounted with pear wax (θ4) were reduced by 14.5 and 65.7% after 8 h incubation, while ΔAaPLC2 and ΔAaPLC3 formed the same infection hyphae as wild type. In addition, AaPLC1 mutation caused pleiotropic effects on fungal biological function, including growth deficiency, changes in stress tolerance, weakening of pathogenicity to the host, as well as destruction of mycotoxin synthesis. Both AaPLC2 and AaPLC3 genes were found to have some effects on stress response and mycotoxin production. Taken together, AaPLC genes differentially regulate the growth, stress response, pathogenicity, and secondary metabolism of A. alternata.

A Basic Guide to the Growth and Manipulation of the Blast Fungus, Magnaporthe oryzae.

The blast fungus, Magnaporthe oryzae, is a devastating plant pathogen that threatens global food security. The social and economic importance of blast disease has contributed to this filamentous fungus becoming a model organism for the study of host‐pathogen interactions. Availability of the complete genome sequences of many strains of the pathogen, as well as rice and wheat cultivars, coupled with the tractability of M. oryzae to classical and molecular genetic manipulation have contributed to its widespread study. Although M. oryzae has been extensively investigated for the past two decades, procedures for storing, maintaining, and manipulating the blast fungus in the laboratory had not been compiled and updated. As a consequence, there is considerable disparity in how the fungus is stored and manipulated between studies. In this article, we present a collection of protocols providing clear explanations of how to preserve filter stocks of M. oryzae; how to grow the fungus in both liquid and solid media; how to extract genomic DNA from fungal mycelium; how to induce appressorium formation on coverslips for visualization and tissue collection; and how to perform two distinct types of plant infection assay for virulence assessment. By sharing our most used laboratory procedures, we aim to address some of the knowledge gaps in current M. oryzae protocols and contribute to uniformity and robustness in studies by the Magnaporthe research community. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Storage of M. oryzae strains Basic Protocol 2: Revival and regular maintenance of M. oryzae cultures in solid medium Alternate Protocol 1: Regular maintenance of M. oryzae cultures in liquid medium Basic Protocol 3: Genomic DNA extraction from M. oryzae mycelium Alternate Protocol 2: Quick DNA extraction from M. oryzae mycelium Basic Protocol 4: M. oryzae induction of appressorium development on glass coverslips for microscopy Alternate Protocol 3: M. oryzae induction of appressorium development on glass coverslips for tissue collection Basic Protocol 5: M. oryzae rice infection assay through spray inoculation Alternate Protocol 4: M. oryzae leaf‐drop plant infection assay

Magnaporthe oryzae Auxiliary Activity Protein MoAa91 Functions as Chitin-Binding Protein To Induce Appressorium Formation on Artificial Inductive Surfaces and Suppress Plant Immunity.

The appressoria that are generated by the rice blast fungus Magnaporthe oryzae in response to surface cues are important for successful colonization. Previous work showed that regulators of G-protein signaling (RGS) and RGS-like proteins play critical roles in appressorium formation. However, the mechanisms by which these proteins orchestrate surface recognition for appressorium induction remain unclear. Here, we performed comparative transcriptomic studies of ΔMorgs mutant and wild-type strains and found that M. oryzae Aa91 (MoAa91), a homolog of the auxiliary activity family 9 protein (Aa9), was required for surface recognition of M. oryzae We found that MoAA91 was regulated by the MoMsn2 transcription factor and that its disruption resulted in defects in both appressorium formation on the artificial inductive surface and full virulence of the pathogen. We further showed that MoAa91 was secreted into the apoplast space and was capable of competing with the immune receptor chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immune responses. In summary, we have found that MoAa91 is a novel signaling molecule regulated by RGS and RGS-like proteins and that MoAa91 not only governs appressorium development and virulence but also functions as an effector to suppress host immunity.IMPORTANCE The rice blast fungus Magnaporthe oryzae generates infection structure appressoria in response to surface cues largely due to functions of signaling molecules, including G-proteins, regulators of G-protein signaling (RGS), mitogen-activated protein (MAP) kinase pathways, cAMP signaling, and TOR signaling pathways. M. oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), and MoRgs1, MoRgs3, MoRgs4, and MoRgs7 were found to be particularly important in appressorium development. To explore the mechanisms by which these proteins regulate appressorium development, we have performed a comparative in planta transcriptomic study and identified an auxiliary activity family 9 protein (Aa9) homolog that we named MoAa91. We showed that MoAa91 was secreted from appressoria and that the recombinant MoAa91 could compete with a chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immunity. By identifying MoAa91 as a novel signaling molecule functioning in appressorium development and an effector in suppressing host immunity, our studies revealed a novel mechanism by which RGS and RGS-like proteins regulate pathogen-host interactions.

Extremely Low Concentrations of Acetic Acid Stimulate Cell Differentiation in Rice Blast Fungus.

SummaryMetabolic switching and rewiring play a dynamic role in programmed cell differentiation. Many pathogenic microbes need to survive in nutrient-deficient conditions and use the glyoxylate cycle, an anaplerotic pathway of the tricarboxylic acid cycle, to produce carbohydrates. The plant pathogenic fungus Magnaporthe oryzae (Pyricularia oryzae) has a unique chitin deacetylase, Cbp1. The spatiotemporal activity of this protein is required for modification of the M. oryzae wall and for cell differentiation into the specialized infection structure (appressorium). Here we show that acetic acid, another product released by the Cbp1-catalyzed conversion of chitin into chitosan, induces appressorium formation. An extremely low concentration (fM) of acetic acid restored cell differentiation in a Δcbp1 mutant possibly through the glyoxylate cycle.

Structure-function analyses of the Pth11 receptor reveal an important role for CFEM motif and redox regulation in rice blast.

The interaction of Magnaporthe oryzae, the rice blast fungus, and rice begins when M.oryzae establishes contact with the host plant surface. On perception of appropriate surface signals, M.oryzae forms appressoria and initiates host invasion. Pth11, an important G-protein-coupled receptor necessary for appressorium formation in M.oryzae, contains seven transmembrane regions and a CFEM (common in several fungal extracellular membrane proteins) domain with the characteristic eight cysteine residues. We focused on gaining further insight into the role of the CFEM domain in the putative surface sensing/response function of Pth11. Increased/constitutive expression of CFEM resulted in precocious, albeit defective, appressoria formation in wild-type M.oryzae. The Pth11C63A/C65A mutant, probably with disrupted disulfide bonds in the CFEM, showed delayed appressorium formation and reduced virulence. Furthermore, the accumulation of reactive oxygen species (ROS) was found to be altered in the pth11Δ strain. Strikingly, antioxidant treatment induced appressorium formation in pth11Δ. The Gα subunit MagB and the mitogen-activated protein (MAP) kinase Pmk1 were required for the formation of antioxidant-induced appressoria. We conclude that the CFEM domain of Pth11 is required for proper development of the appressoria, appressoria-like structures and pathogenicity. Highly regulated ROS homeostasis is important for Pth11-mediated appressorium formation in M.oryzae.

GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling.

Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development.

Differences between appressoria formed by germ tubes and appressorium-like structures developed by hyphal tips in Magnaporthe oryzae

Melanized appressoria are highly specialized infection structures formed by germ tubes of the rice blast fungus Magnaporthe oryzae for plant infection. M. oryzae also forms appressorium-like structures on hyphal tips. Whereas appressorium formation by conidial germ tubes has been well characterized, formation of appressorium-like structures by hyphal tips is under-investigated. In a previous study, we found that the chs7 deletion mutant failed to form appressoria on germ tubes but were normal in the development of appressorium-like structures on artificial hydrophobic surfaces. In this study, we compared the differences between the formation of appressoria by germ tubes and appressorium-like structures by hyphal tips in M. oryzae. Structurally, both appressoria and appressorium-like structures had a melanin layer that was absent in the pore region. In general, the latters were 1.4-fold larger in size but had lower turgor pressure than appressoria, which is consistent with its lower efficiency in plant penetration. Treatments with cAMP, IBMX, or a cutin monomer efficiently induced appressorium formation but not the development of appressorium-like structures. In contrast, coating surfaces with waxes stimulated the formation of both infection structures. Studies with various signaling mutants indicate that Osm1 and Mps1 are dispensable but Pmk1 is essential for both appressorium formation and development of appressorium-like structures on hyphal tips. Interestingly, the cpkA mutant was reduced in the differentiation of appressorium-like structures but not appressorium formation. We also observed that the con7 mutant generated in our lab failed to form appressorium-like structures on hyphal tips but still produced appressoria by germ tubes on hydrophobic surfaces. Con7 is a transcription factor regulating the expression of CHS7. Overall, these results indicate that the development of appressorium-like structures by hyphal tips and formation of appressoria by germ tubes are not identical differentiation processes in M. oryzae and may involve different molecular mechanisms.

Calcium/calmodulin-dependent signaling for prepenetration development in Cochliobolus miyabeanus infecting rice

Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect rice. Contacting a hard surface induces appressorium formation in C. miyabeanus, while the hydrophobicity of the substratum does not affect this morphogenic infection event. To determine whether the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. miyabeanus, the effects of a calcium chelator (ethylene glycol tetraacetic acid; EGTA), phospholipase C inhibitor (neomycin), intracellular calcium channel blocker (TMB-8), calmodulin antagonists (chlorpromazine, phenoxybenzamine, and W-7), and calcineurin inhibitor (cyclosporin A) on morphogenesis and infection were examined. Addition of Ca2+ and the calcium ionophore A23187 did not affect conidial germination, while the number of appressoria decreased with higher concentrations. EGTA inhibited conidial germination and appressorium formation. The calcium channel blocker did not affect appressorium formation at any concentration; however, calmodulin antagonists and the calcineurin inhibitor specifically reduced appressorium formation at the micromolar level. One of the calmodulin antagonists, W-7, also inhibited accumulation of mRNA of the calmodulin gene within germinating conidia and/or appressorium-forming germ tubes. Thus, biochemical processes controlled by the calcium/calmodulin signaling system seem to be involved in the induction of prepenetration morphogenesis on rice.

A Gγ subunit promoter T-DNA insertion mutant—A1-412 of Magnaporthe grisea is defective in appressorium formation, penetration and pathogenicity

In our previous studies, a nonpathogenic mutant of Magnaporthe grisea, A1-412, which was defective in appressorium formation, penetration and infectious growth, was obtained by T-DNA insertional mutagenesis. Here we reported the identification and characterization of the corresponding gene. Sequence analysis of the genomic DNA franking T-DNA isolated by TAIL-PCR technique showed that T-DNA was inserted into the promoter region of the predicted G protein γ-subunit gene MGG1 (for Magnaporthe grisea G protein Gamma subunit). MGG1 is predicted to encode a 93-aa protein with a typical G-protein gamma like domain (GGL) and C-terminal CAAX box. The amino-acid sequence of MGG1 is highly identical to the Gγ subunits of other filamentous fungi. Further phenotypic investigation of A1-412 showed that exogenous cAMP could induce appressorium formation, although the formed appressoria were abnormal in shape and unable to penetrate onion epidemis or rice leaves. Moreover, few perithecia were observed when A1-412 was crossed with the appropriate mating-type strain. The above phenotypes in A1-412 were partially complemented by reintroduction of the gene MGG1. Our results indicate that the G-protein gamma subunit MGG1 may be involved in regulating morphogenesis, mating and pathogenicity in M. grisea.

Signal transduction leading to appressorium formation in germinating conidia of Magnaporthe grisea: effects of second messengers diacylglycerols, ceramides and sphingomyelin

The inhibition of appressorium formation in germinating conidiospores of Magnaporthe grisea on an inductive surface by glisoprenin A could be reversed in a competitive manner by 1,2-dioctanoylglycerol, a known activator of protein kinase C. Dioctanoylglycerol and linoleic acid methyl ester induced appressorium formation on a noninductive surface. The effect of ceramides as inducers was heavily dependent on the fatty acid in the molecule. Ceramide IIIa containing linoleic acid was a good inducer whereas others had only weak effects. Sphingomyelin, however, inhibited appressorium formation induced by 1,16-hexadecanediol and to a lesser extent after induction with 8-(4-chlorophenylthio)-adenosine-3′,5′-monophosphate. The results are in agreement with our previous findings that in appressorium formation two signal transducing pathways are involved.

Signaling pathways involved in preinfection development of Colletotrichum gloeosporioides, C. coccodes, and C. dematium pathogenic on red pepper

Colletotrichum gloeosporioides, C. coccodes, and C. dematium are responsible for pepper anthracnose and they infect pepper as tissue-specific manner. They form a specialized infection structure, an appressorium, to infect the host. Contacting hard surface induces appressorium formation in these fungi, whereas hydrophobicity of the contact surface does not affect this infection-related differentiation. To investigate the common signal transduction pathways involved in conidial germination and appressorium formation, several pharmacological agents thought to act on critical steps in cAMP- and calcium-dependent signaling pathways were tested. Exogenous addition of cAMP did not alter both differentiations of all 3 anthracnose pathogens tested. Two phosphodiesterase inhibitors, caffeine and isobutylmethylxanthine (IBMX), specifically diminished appressorium formation of C. gloeosporioides and conidial germination of C. dematium. Addition of Ca 2+ specifically reduced appressorium formation of C. coccodes and C. dematium, whereas EGTA (calcium chelator) and neomycin (phospholipase C inhibitor) showed nonspecific inhibitory effects on both developments. Calcium channel blockers and calmodulin antagonists showed species-specific inhibitory effects. These results suggest that calcium/calmodulin-dependent signaling pathway plays an essential role on conidial germination and appressorium formation in pepper anthracnose pathogens.

Serial Analysis of Gene Expression (SAGE) of Magnaporthe grisea: genes involved in appressorium formation.

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.

The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Magnaporthe grisea.

Trimeric G-proteins transmit extracellular signals to various downstream effectors (e.g. MAP kinases) in eukaryotes. In the rice blast fungus Magnaporthe grisea, the Pmk1 MAP kinase is essential for appressorium formation and infectious growth. The pmk1 deletion mutant fails to form appressoria but still responds to exogenous cAMP for tip deformation. Since gene disruption mutants of three Galpha subunits still form appressoria and are phenotypically different from pmk1 mutants, it is likely that the Pmk1 pathway is activated by Gbeta in M. grisea. In this study, we isolated and characterized the MGB1 gene that encodes the G subunit in M. grisea. Mutants disrupted in MGB1 were reduced in conidiation. Conidia from mgb1 mutants were defective in appressorium formation and failed to penetrate or grow invasively on rice leaves. Exogenous cAMP induced appressorium formation in mgb1 mutants, but these appressoria were abnormal in shape and could not penetrate. The intracellular cAMP level was reduced in mgb1 mutants and the defects in conidiation and hyphal growth were partially suppressed with 1 mM cAMP. Transformants expressing multiple copies of MGB1 were able to form appressoria on hydrophilic surfaces. Our results suggest that MGB1 may be involved in the cAMP signalling for regulating conidiation, surface recognition and appressorium formation. The Pmk1 pathway may be the downstream target of MGB1 for regulating penetration and infectious hyphae growth in M. grisea.

Calcium/Calmodulin-Dependent Signaling for Prepenetration Development inColletotrichum gloeosporioides

ABSTRACT Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, for host infection. Contacting hard surface induces appressorium formation in C. gloeosporioides, whereas hydrophobicity of the contact surface does not affect this infection-related differentiation. To determine if the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper, effects of calcium chelator (EGTA), phospholipase C inhibitor (neomycin), intracellular calcium modulators (TMB-8 and methoxy verampamil), and calmodulin antagonists (chloroproma-zine, phenoxy benzamine, and W-7) were tested on conidial germination and appressorium formation. Exogenous addition of Ca(2+), regardless of concentration, augmented conidial germination, while appressorial differentiation decreased at higher concentrations. Inhibition of appressorium formation by EGTA was partly restored by the addition of calcium ionophore A23187 or CaCl(2). Calcium channel blockers and calmodulin antagonists specifically reduced appressorium formation at micromolar levels. These results suggest that biochemical processes controlled by the calcium/calmodulin signaling system are involved in the induction of prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper.

Cyclic AMP Restores Appressorium Formation Inhibited by Polyamines in Magnaporthe grisea

ABSTRACT Magnaporthe grisea, the causal agent of rice blast, forms a dome-shaped melanized infection structure, an appressorium, to infect its host. Environmental cues that induce appressorium formation in this fungus include the hydrophobicity and hardness of the contact surface and chemicals produced by the host. An elevated concentration of intracellular cyclic AMP (cAMP) has been implicated in appressorium differentiation in M. grisea. Polyamines (putrescine, spermidine, and sper-mine) are involved in cell growth and differentiation in a wide range of organisms. To understand the role of polyamines in appressorium differentiation in M. grisea, intracellular polyamines were quantified, and the effects of polyamines and polyamine biosynthesis inhibitors on conidial germination and appressorium formation were tested. High levels of polyamines were detected in freshly collected spores, but the levels decreased during conidial germination. Spermidine was found to be the major component. Polyamines and polyamine biosynthesis inhibitors did not affect conidial germination, but polyamines specifically impaired appressorium formation. Furthermore, exogenous addition of cAMP restored appressorium formation inhibited by poly-amines. These results suggest that polyamines may reduce intracellular cAMP levels in M. grisea, leading to the inhibition of appressorium formation.

Signal transduction leading to appressorium formation in germinating conidia of Magnaporthe grisea: effects of second messengers diacylglycerols, ceramides and sphingomyelin

The inhibition of appressorium formation in germinating conidiospores of Magnaporthe grisea on an inductive surface by glisoprenin A could be reversed in a competitive manner by 1,2-dioctanoylglycerol, a known activator of protein kinase C. Dioctanoylglycerol and linoleic acid methyl ester induced appressorium formation on a noninductive surface. The effect of ceramides as inducers was heavily dependent on the fatty acid in the molecule. Ceramide IIIa containing linoleic acid was a good inducer whereas others had only weak effects. Sphingomyelin, however, inhibited appressorium formation induced by 1,16-hexadecanediol and to a lesser extent after induction with 8-(4-chlorophenylthio)-adenosine-3′,5′-monophosphate. The results are in agreement with our previous findings that in appressorium formation two signal transducing pathways are involved.

Cuticular waxes relieve self-inhibition of germination and appressorium formation by the conidia ofMagnaporthe grisea

We tested whether germination and appressorium formation by the conidia ofMagnaporthe grisearequires a hydrophobic surface. On specially cleaned cover glass surfaces conidia ofM. griseagerminated but did not form appressoria. Addition of rice leaf wax, other plant surface waxes or syntheticn-C22fatty acid, fatty alcohol or alkane induced appressorium formation. The requirement for the presence of hydrophobic material to induce appressorium formation was dependent on the conidial population density. As the conidial concentration decreased from 105ml−1to 104ml−1, the need for the lipid decreased to the point of not requiring any lipid to obtain nearly complete appressorium formation, suggesting possible presence of self-inhibitors on the conidia. This suggestion was supported by the observation that the surface lipids washed out of the conidia inhibited germination and appressorium formation in a dose-dependent manner. This inhibition could be reversed by plant surface wax. These results suggest that conidia ofM. griseacarry lipophilic self-inhibitors which prevent germination at the site of sporulation, and that the plant cuticle can relieve this self-inhibition when conidia land on the plant surface.

Chemical signals responsible for appressorium formation in the rice blast fungus Magnaporthe grisea

Magnaporthe grisea,the causative agent of rice blast disease, penetrates the host cuticle directly by way of a highly melanized attachment cell called an appressorium. The signals responsible for appressorium formation are not well known. We report on the sufficiency of a hydrophobic surface to induce appressorium formation in vitrofor a collection of M. griseaisolates. Chemical cues, similar to those available on the host surface, were also found to induce appressorium formation selectively using a novel in vitroassay. The plant cutin monomers cis-9,10-epoxy-18-hydroxyoctadecanoic acid and cis-9-octadecen-1-ol were found to induce infection structure formation in the nanomolar range, while other monomers were effective only at the micromolar range. No differences were detected for appressorium formation rates on various host and non-host plants suggesting appressorium initiation is not host specific. Plant lipid and wax compounds were also tested; the two most effective inducers were found to be 1,16-hexadecanedial and 1,16-hexadecanediol which had AID 50s (chemical dose at which appressoria induction equals 50% for germinated conidia) of 3.86n mand 7.25n mrespectively. Structural studies of the inducing compounds were performed, and terminal hydrogen bonds and chain length were found to be important for high activity. The thigmotropic effects and chemical cues are discussed in the context of possible mechanisms involved and a working model of infection related morphogenesis of the plant surface is presented.

Inhibition of thigmostimulated cell differentiation with RGD-peptides inUromyces germlings

Germlings of the plant pathogenic fungusUromyces appendiculatus sense and respond to topographical signals of various substrata by undergoing a cell differentiation process that culminates in a structure termed an appressorium. In some cell systems, recognition and mediation of extracellular signals is via transmembrane glycoproteins known as integrins that often exhibit specific affinities to the tripeptide sequence Arg-Gly-Asp (RGD) found in several extracellular matrix components. Germlings grown on substrata inductive for appressorium formation in the presence of buffered synthetic peptides containing the amino acid sequence RGD, e.g., RGD, RGDS, GRGD, and GRGDGSPK (0.5–2.0 mM), were inhibited from developing appressoria. Two non-RGD peptides (GGGG and RGES) as well as two RGD peptides (GRGDS and RGDSPASSKP) did not inhibit appressorium formation. Germling growth was not significantly affected by any of the peptides. Furthermore, 0.5 μm diameter micropipettes that are normally inductive for appressorium formation when positioned between the germling apex and the substratum did not induce appressorium formation when coated with the RGD peptide. Silanized micropipettes left uncoated or coated with RGES were inductive for appressorium formation. Those observations lead to the hypothesis that an integrin-like protein may be involved in the process of signaling for initiation of appressorium formation inUromyces. An RGDSPC-affinity column was used to isolate proteins fromUromyces germlings with affinity to the RGD sequence. Elution with RGD or EDTA, but not with RGES, yielded at least 12 proteins of which one protein (95 kDa) expressed affinity on immunoblots to two different antibodies of β1-integrin; one to the carboxyl-terminus of a synthetic peptide of integrin from chicken, and the other from the amino terminus of integrin from human placenta.