Two novel genes induced by hard-surface contact of Colletotrichum gloeosporioides conidia

Germinating conidia of many phytopathogenic fungi must differentiate into an infection structure called the appressorium in order to penetrate into their hosts. This differentiation is known to require contact with a hard surface. However, the molecular basis for this requirement is not known. Induction of this differentiation in the avocado pathogen, Colletotrichum gloeosporioides, by chemical signals such as the host's surface wax or the fruit-ripening hormone, ethylene, requires contact of the conidia with a hard surface for about 2 h. To study molecular events triggered by hard-surface contact, we isolated several genes expressed during the early stage of hard-surface treatment by a differential-display method. The genes that encode Colletotrichum hard-surface induced proteins are designated chip genes. In this study, we report the characterization of CHIP2 and CHIP3 genes that would encode proteins with molecular masses of 65 and 64 kDa, respectively, that have no homology to any known proteins. The CHIP2 product would contain a putative nuclear localization signal, a leucine zipper motif, and a heptad repeat region which might dimerize into coiled-coil structure. The CHIP3 product would be a nine-transmembrane-domain-containing protein. RNA blots showed that CHIP2 and CHIP3 are induced by a 2-h hard-surface contact. However, disruption of these genes did not affect the appressorium-forming ability and did not cause a significant decrease in virulence on avocado or tomato fruits suggesting that C. gloeosporioides might have genes functionally redundant to CHIP2 and CHIP3 or that these genes induced by hard-surface contact control processes not directly involved in pathogenesis.     

Identification of a Gene Product Induced by Hard-Surface Contact of Colletotrichum gloeosporioides Conidia as a Ubiquitin-Conjugating Enzyme by Yeast Complementation

The germinating conidia of many phytopathogenic fungi on hosts must differentiate into an infection structure called the appressorium in order to penetrate their hosts. Chemical signals, such as the host’s surface wax or fruit ripening hormone, ethylene, trigger germination and appressorium formation of the avocado pathogen Colletotrichum gloeosporioides only after the conidia are in contact with a hard surface. What role this contact plays is unknown. Here, we describe isolation of genes expressed during the early stage of hard-surface treatment by a differential-display method and report characterization of one of these cloned genes, chip1 (Colletotrichum hard-surface induced protein 1 gene), which encodes a ubiquitin-conjugating enzyme. RNA blots clearly showed that it is induced by hard-surface contact and that ethylene treatment enhanced this induction. The predicted open reading frame (ubc1_Cg) would encode a 16.2-kDa ubiquitin-conjugating enzyme, which shows 82% identity to the Saccharomyces cerevisiae UBC4-UBC5 E2 enzyme, comprising a major part of total ubiquitin-conjugating activity in stressed yeast cells. UBC1_Cg can complement the proteolysis deficiency of the S. cerevisiae ubc4 ubc5 mutant, indicating that ubiquitin-dependent protein degradation is involved in conidial germination and appressorial differentiation.     

Chemical Signals from Avocado Surface Wax Trigger Germination and Appressorium Formation in Colletotrichum gloeosporioides

The surface wax of the host, avocado (Persea americana) fruit, induced germination and appressorium formation in the spores of Colletotrichum gloeosporioides. Waxes from nonhost plants did not induce appressorium formation in this fungus, and avocado wax did not induce appressorium formation in most Colletotrichum species that infect other hosts. Bioassays of the thin-layer chromatographic fractions of the avocado wax showed that the fatty alcohol fraction was the main appressorium-inducing component. Testing of authentic n-C8 to n-C32 fatty alcohols revealed that C24 and longer-chain alcohols induced appressorium formation. Gas-liquid chromatography/mass spectrometry analysis of free fatty alcohols revealed that avocado wax contains a high content of very long chains. Waxes from nonhost plants containing an even higher content of the very long-chain alcohols did not induce appressorium formation. Waxes from nonhost plants strongly inhibited appressorium induction by avocado wax. Thus, a favorable balance between appressorium-inducing very long-chain fatty alcohols and the absence of inhibitors allows the fungus to use the host surface wax to trigger germination and differentiation of infection structures in the pathogen.     

Identification of a hard surface contact-induced gene in Colletotrichum gloeosporioides conidia as a sterol glycosyl transferase,a novel fungal virulence factor

Hard surface contact has been known to be necessary to induce infection structure (appressorium) formation in many phytopathogenic fungi. However, the molecular basis of this requirement is unknown. We have used a differential display approach to clone some of the genes induced in the conidia by hard surface contact. We report that one of the genes induced by hard-surface contact of the conidia of Colletotrichum gloeosporioides, chip6, encodes a protein with homology to sterol glycosyl transferases. chip6 expressed in E. coli catalyses glucosyl transfer from UDP-glucose to cholesterol. Disruption of chip6 causes a marked decrease in the transferase activity and a drastic reduction in virulence on its natural host, avocado fruits, although the mutant is capable of normal growth and appressorium formation. The requirement for sterol glycosyl transferase for pathogenicity suggests a novel biological function for this transferase.     

Participation of Oryza sativa leaf wax in appressorium formation by Pyricularia oryzae

Appressorium formation of Pyricularia oryzae P2 on cover-glass coated with each of the components of rice leaf wax was examined. Wax esters, aldehydes and alcohols, having polar groups and low contact angles, promoted appressorium formation, but alkanes, non-polar molecules having high contact angles, had no effects. Germination of conidia, however, was not aftected with those constituents.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

The Cyclase-Associated Protein Cap1 Is Important for Proper Regulation of Infection-Related Morphogenesis in Magnaporthe oryzae

Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1 ^ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1 ^ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M. oryzae. CAP1 may also play a role in feedback inhibition of Ras2 signaling when Pmk1 is activated.     

Perilipin LDP1 coordinates lipid droplets formation and utilization for appressorium-mediated infection in Magnaporthe oryzae

Lipid droplets (LDs) serve as one of the major reservoirs in conidia of Magnaporthe oryzae and are quickly utilized during appressorium formation. Here, we identified a gene, LDP1, encoding a perilipin that is important for LD formation and utilization during appressorium maturation. LDP1 is highly expressed in conidium and immature appressorium. Disruption mutants of LDP1 were significantly reduced in virulence, due to appressorial turgor reduction and difficulty in penetration. LDs were significantly reduced in the Δldp1 mutant, indicating LDP1 was required for LDs formation. LDP1 was colocalized with the LDs in conidium and immature appressorium but was gradually separated during appressorium maturation. A typical intracellular triacylglycerol lipase, TGL1-2, was clearly separated with LDs in conidium and immature appressorium but was well colocalized with LDs during appressorium maturation. The subcellular localization of TGL1-2 was affected by LDP1. These data suggested that LDP1 was bound to LDs for protecting from utilization in conidia and at the early appressorium stage but was separated from LDs for lipase entering and degradation. LDP1 was phosphorylated by CPKA at Thr96, which was essential for its localization and functions. These data indicate perilipin LDP1 can coordinate LD formation and utilization for appressorium-mediated infection of M. oryzae.     

Plasmid Encoded AcrAB–TolC Tripartite Multidrug-Efflux System in Acidiphilium symbioticum H8

Acidophilic bacterium, Acidiphilium symbioticum H8, is resistant to high levels of several heavy metals, hydrophobic agents, and organic solvents. The ~9.6 kb plasmid pASH8, was purified, digested with HindIII, and sub-cloned in pUC19 at the respective site. Three different fragment size clones were achieved. The clones were completely sequenced and analyzed. The first clone encodes for a single putative open reading frame (ORF), which showed significant homology to several rusticyaninA1 proteins. The second clone encodes for a 43-kDa protein, which has conserved domain homology with several outer envelop TolC proteins. The clone with pASH8 tolC gene can functionally complement an Escherichia coli tolC mutant strain, making it resistant to several toxic hydrophobic agents, earlier for which it was sensitive. The tolC gene was found to be essential for imparting resistance to the clone toward these toxic hydrophobic agents. The third clone encodes for a putative 318-aa AcrA (acriflavine resistance protein A) protein and the clone was resistance to plasmid curing dye acriflavine. The clone also has a truncated ORF, which showed significant homology to cation-efflux pump AcrB. This study is the first to report a multi-drug efflux system to be encoded on a plasmid of any Acidiphilium strain.