Many plant pathogenic fungi, such as the cereal pathogen Colletotrichum graminicola, differentiate highly specialized infection structures called appressoria, which send a penetration peg into the ...underlying plant cell. Appressoria have been shown to generate enormous turgor pressure, but direct evidence for mechanical infection of plants by fungi is lacking. A microscopic method was developed that uses elastic optical waveguides to visualize and measure forces locally exerted by single appressoria. By this method, the force exerted by appressoria of C. graminicola was found to be about 17 micronewtons.
Small Ras superfamily GTPases are highly conserved regulatory factors of fungal cell wall biosynthesis and morphogenesis. Previous experiments have shown that the Rho4-like protein of the maize ...anthracnose fungus Colletotrichum graminicola, formerly erroneously annotated as a Rho1 protein, physically interacts with the β-1,3-glucan synthase Gls1 (Lange et al., 2014; Curr. Genet. 60:343–350). Here, we show that Rho4 is required for β-1,3-glucan synthesis. Accordingly, Δrho4 strains formed distorted vegetative hyphae with swellings, and exhibited strongly reduced rates of hyphal growth and defects in asexual sporulation. Moreover, on host cuticles, conidia of Δrho4 strains formed long hyphae with hyphopodia, rather than short germ tubes with appressoria. Hyphopodia of Δrho4 strains exhibited penetration defects and often germinated laterally, indicative of cell wall weaknesses. In planta differentiated infection hyphae of Δrho4 strains were fringy, and anthracnose disease symptoms caused by these strains on intact and wounded maize leaf segments were significantly weaker than those caused by the WT strain. A retarded disease symptom development was confirmed by qPCR analyses. Collectively, we identified the Ras GTPase Rho4 as a new virulence factor of C. graminicola.
Typically, pathogenesis of the hemibiotroph Colletotrichum graminicola and defense responses of its host, Zea mays, are studied on young leaves. Equivalent studies have not been performed with leaves ...undergoing senescence, a situation that is relevant in the field. We discovered that, in contrast to anthracnose symptoms formed on young and mature leaves, green islands reminiscent of those known from obligate biotrophs were formed on senescing leaves. Microscopy revealed that the fungus grew in both symptoms from the epidermis towards the bundle sheath. In green islands, tissues remained intact for an extended time period. Imaging PAM (pulse-amplitude-modulation) fluorescence analyses revealed that photosynthesis is transiently maintained at green islands but declined in tissue surrounding the infection. In younger leaves however, photosynthesis was reduced only at infection sites. Support for the local modification of host physiology came from quantitative reverse transcription-polymerase chain reaction analyzing gene expression at high spatial resolution. Decreased transcript levels of the senescence markers see1 and ccp1 corroborated a pathogen-induced delay of senescence. Expression of several genes encoding proteins involved in photosynthesis was strongly reduced by infection. In contrast, transcript levels of incw1, encoding a cell-wall invertase, were increased 70-fold at green islands, suggesting that C. graminicola induced carbon sinks in senescing tissue.
Penetration attempts of the hemibiotroph Colletotrichum graminicola may activate PAMP-triggered immunity (PTI) on different cultivars of Zea mays to different extent. However, in most events, this ...does not prevent the establishment of a compatible pathogenic interaction. In this study, we investigate the extent to which the host variety influences PTI. Furthermore, we assess whether visual disease symptoms occurring on different maize varieties reliably reflect fungal biomass development in planta as determined by qPCR and GFP tracing.
Employing a set of four maize varieties, which were selected from a panel of 27 varieties, for in-depth assessment of pathogenesis of the wild type strain of C. graminicola, revealed considerable differences in susceptibility as evidenced by symptom severity that decreased from variety Golden Jubilee to Mikado to Farmtop to B73. However, a newly developed qPCR assay and microscopical observation of a GFP-labelled strain showed that disease symptoms are in some instances inconsistent when compared with other indicators of susceptibility. Of the four varieties assessed, either Golden Jubilee, Mikado and B73, or Golden Jubilee, Farmtop and B73 showed a direct correlation between symptom and fungal biomass development. In a pairwise comparison, however, Mikado and Farmtop showed an inverse correlation for these features.
The genotype of maize contributes to the severity of symptoms resulting from an infection with C. graminicola. Partially, this may be attributed to the extent of PTI activated in different varieties, as reflected by papilla formation. Furthermore, when evaluating the susceptibility of a variety, it should be considered that symptom severity must not have to reflect the extent of fungal growth in the infected tissue.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on Zea mays. Following penetration of epidermal cells, the fungus enters a short biotrophic phase, ...followed by a destructive necrotrophic phase of pathogenesis. During both phases, secreted fungal proteins are supposed to determine progress and success of the infection. To identify genes encoding such proteins, we constructed a yeast signal sequence trap (YSST) cDNA-library from RNA extracted from mycelium grown in vitro on corn cell walls and leaf extract. Of the 103 identified unigenes, 50 showed significant similarities to genes with a reported function, 25 sequences were similar to genes without a known function, and 28 sequences showed no similarity to entries in the databases. Macroarray hybridization and quantitative reverse-transcriptase polymerase chain reaction confirmed that most genes identified by the YSST screen are expressed in planta. Other than some genes that were constantly expressed, a larger set showed peaks of transcript abundances at specific phases of pathogenesis. Another set exhibited biphasic expression with peaks at the biotrophic and necrotrophic phase. Transcript analyses of in vitro-grown cultures revealed that several of the genes identified by the YSST screen were induced by the addition of corn leaf components, indicating that host-derived factors may have mimicked the host milieu.
To elucidate the function of a protein, it is crucial to know its subcellular location and its interaction partners. Common approaches to resolve those questions rely on the genetic tagging of the ...gene-of-interest (GOI) with fluorescent reporters. To determine the location of a tagged protein, it may be co-localized with tagged marker proteins. The interaction of two proteins under investigation is often analysed by tagging both with the C- and N-terminal halves of a fluorescent protein. In fungi, the tagged GOI are commonly introduced by serial transformation with plasmids harbouring a single tagged GOI and subsequent selection of suitable strains. In this study, a plasmid system is presented that allows the tagging of several GOI on a single plasmid. This novel double tagging plasmid system (DTPS) allows a much faster and less laborious generation of double-labelled fungal strains when compared with conventional approaches. The DTPS also enables the combination of as many tagged GOI as desired and a simple exchange of existing tags. Furthermore, new tags can be introduced smoothly into the system. In conclusion, the DTPS allows an efficient tagging of GOI with a high degree of flexibility and therefore accelerates functional analysis of proteins in vivo.
In filamentous fungi, Sfp-type 4'-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary (α-aminoadipate reductase AAR) and secondary (polyketide synthases and nonribosomal ...peptide synthetases) metabolism. We cloned the PPTase gene PPT1 of the maize anthracnose fungus Colletotrichum graminicola and generated PPTase-deficient mutants (Δppt1). Δppt1 strains were auxotrophic for Lys, unable to synthesize siderophores, hypersensitive to reactive oxygen species, and unable to synthesize polyketides (PKs). A differential analysis of secondary metabolites produced by wild-type and Δppt1 strains led to the identification of six novel PKs. Infection-related morphogenesis was affected in Δppt1 strains. Rarely formed appressoria of Δppt1 strains were nonmelanized and ruptured on intact plant. The hyphae of Δppt1 strains colonized wounded maize (Zea mays) leaves but failed to generate necrotic anthracnose disease symptoms and were defective in asexual sporulation. To analyze the pleiotropic pathogenicity phenotype, we generated AAR-deficient mutants (Δaar1) and employed a melanin-deficient mutant (M1.502). Results indicated that PPT1 activates enzymes required at defined stages of infection. Melanization is required for cell wall rigidity and appressorium function, and Lys supplied by the AAR1 pathway is essential for necrotrophic development. As PPTase-deficient mutants of Magnaporthe oryzea were also nonpathogenic, we conclude that PPTases represent a novel fungal pathogenicity factor.
Summary
The hemibiotrophic maize pathogen
C
olletotrichum graminicola
synthesizes one intracellular and three secreted siderophores.
eGFP
fusions with the key siderophore biosynthesis gene,
SID1
, ...encoding
l
‐ornithine‐
N
5
‐monooxygenase, suggested that siderophore biosynthesis is rigorously downregulated specifically during biotrophic development. In order to investigate the role of siderophores during vegetative development and pathogenesis,
SID1
, which is required for synthesis of all siderophores, and the non‐ribosomal peptide synthetase gene
NPS6
, synthesizing secreted siderophores, were deleted. Mutant analyses revealed that siderophores are required for vegetative growth under iron‐limiting conditions, conidiation,
ROS
tolerance, and cell wall integrity. Δ
sid1
and Δ
nps6
mutants were hampered in formation of melanized appressoria and impaired in virulence. In agreement with biotrophy‐specific downregulation of siderophore biosynthesis, Δ
sid1
and Δ
nps6
strains were not affected in biotrophic development, but spread of necrotrophic hyphae was reduced. To address the question why siderophore biosynthesis is specifically downregulated in biotrophic hyphae, maize leaves were infiltrated with siderophores. Siderophore infiltration alone did not induce defence responses, but formation of biotrophic hyphae in siderophore‐infiltrated leaves caused dramatically increased
ROS
formation and transcriptional activation of genes encoding defence‐related peroxidases and
PR
proteins. These data suggest that fungal siderophores modulate the plant immune system.
Plant pathogenic fungi differentiate a highly specialized infection cell called an appressorium to infect their hosts. Appressorium formation is induced by specific physical or chemical cues provided ...by the host plant. Several components of signal transduction pathways have been identified that are involved in infection-related morphogenesis and virulence or pathogenicity. In this review we summarize current knowledge on appressorium differentiation and function, and discuss aspects of initial stages of fungal development in the plant.
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