The pheromone response factor coordinates filamentous growth and pathogenicity in Ustilago maydis.

In Ustilago maydis, the a and b mating type loci regulate cell fusion, filamentous growth and pathogenicity. The a locus encodes a pheromone-based cell recognition system, and the b locus specifies two homeodomain proteins. The expression of all genes in the a and b loci is induced by pheromone. We have identified a HMG protein (Prf1) that binds sequence specifically to pheromone response elements present in the a and b loci. prf1 mutants do not express the a and b genes and are sterile. The disruption of prf1 in pathogenic haploid strains results in a loss of pathogenicity. The constitutive expression of the b genes restores pathogenicity and induces filamentous growth in the absence of the pheromone signal. These results provide evidence that pheromone signalling, filamentous growth and pathogenic development are linked through Prf1.     

The Gbeta-subunit-encoding gene bpp1 controls cyclic-AMP signaling in Ustilago maydis

In the phytopathogenic fungus Ustilago maydis, fusion of haploid cells is a prerequisite for infection. This process is controlled by a pheromone-receptor system. The receptors belong to the seven-transmembrane class that are coupled to heterotrimeric G proteins. Of four Galpha subunits in U. maydis, only gpa3 has a function during mating and cyclic AMP (cAMP) signaling. Activation of the cAMP cascade induces pheromone gene expression; however, it does not lead to the induction of conjugation tubes seen after pheromone stimulation. To investigate the possibility that a Gbeta subunit participates in pheromone signaling, we isolated the single beta subunit gene, bpp1, from U. maydis. bpp1 deletion mutants grew filamentously and showed attenuated pheromone gene expression, phenotypes associated with deltagpa3 strains. In addition, a constitutively active allele of gpa3 suppressed the phenotype of the bpp1 deletion strains. We suggest that Bpp1 and Gpa3 are components of the same heterotrimeric G protein acting on adenylyl cyclase. Interestingly, while deltagpa3 strains are impaired in pathogenicity, deltabpp1 mutants are able to induce plant tumors. This could indicate that Gpa3 operates independently of Bpp1 during pathogenic development.     

A pheromone receptor gene, pre-1, is essential for mating type-specific directional growth and fusion of trichogynes and female fertility in Neurospora crassa

Neurospora crassa is a heterothallic filamentous fungus with two mating types, mat a and mat A. Its mating involves differentiation of female reproductive structures (protoperithecia) and chemotropic growth of female-specific hyphae (trichogynes) towards a cell of the opposite mating type in a pheromone-mediated process. In this study, we characterize the pre-1 gene, encoding a predicted G-protein-coupled receptor with sequence similarity to fungal pheromone receptors. pre-1 is most highly expressed in mat A strains under mating conditions, but low levels can also be detected in mat a strains. Analysis of pre-1 deletion mutants showed that loss of pre-1 does not greatly affect vegetative growth, heterokaryon formation or male fertility in either mating type. Protoperithecia from Deltapre-1 mat A mutants do not undergo fertilization; this defect largely stems from an inability of their trichogynes to recognize and fuse with mat a cells. Previous work has demonstrated that the Galpha subunit, GNA-1, and the Gbeta protein, GNB-1, are essential for female fertility in N. crassa. Trichogynes of Deltagna-1 and Deltagnb-1 mutants displayed severe defects in growth towards and fusion with male cells, similar to that of Deltapre-1 mat A strains. However, the female sterility defect of the Deltapre-1 mat A mutant could not be complemented by constitutive activation of gna-1, suggesting additional layers of regulation. We propose that PRE-1 is a pheromone receptor coupled to GNA-1 that is essential for the mating of mat A strains as females, consistent with a role in launching the pheromone response pathway in N. crassa.     

Tomato mutants altered in bacterial disease resistance provide evidence for a new locus controlling pathogen recognition.

We have employed a genetic approach to study the resistance of tomato to the phytopathogenic bacterium Pseudomonas syringae pv tomato. Resistance to P. s. tomato depends upon expression of the Pto locus in tomato, which encodes a protein with similarity to serine/threonine protein kinases and recognizes pathogen strains expressing the avirulence gene avrPto. Eleven tomato mutants were isolated with altered resistance to P. s. tomato strains expressing avrPto. We identified mutations both in the Pto resistance locus and in a new locus designated Prf (for Pseudomonas resistance and fenthion sensitivity). The genetic approach allowed us to dissect the roles of these loci in signal transduction in response to pathogen attack. Lines carrying mutations in the Pto locus vary 200-fold in the degree to which they are susceptible to P. s. tomato strains expressing avrPto. The pto mutants retain sensitivity to the organophosphate insecticide fenthion; this trait segregates with Pto in genetic crosses. This result suggested that contrary to previous hypotheses, the Pto locus controls pathogen recognition but not fenthion sensitivity. Interestingly, mutations in the prf locus result in both complete susceptibility to P. s. tomato and insensitivity to fenthion, suggesting that Prf plays a role in tomato signaling in response to both pathogen elicitors and fenthion. Because pto and prf mutations do not alter recognition of Xanthomonas campestris strains expressing avrBsP, an avirulence gene recognized by all tested tomato cultivars, Prf does not play a general role in disease resistance but possibly functions specifically in resistance against P. s. tomato. Genetic analysis of F2 populations from crosses of pto and prf homozygotes indicated that the Pto and Prf loci are tightly linked.     

A Mads-Box Homologue in Ustilago Maydis Regulates the Expression of Pheromone-Inducible Genes but Is Nonessential

Mating and pathogenic development in the smut fungus Ustilago maydis are controlled by a pheromone/receptor system and two homeodomain proteins, bEp and bWp, which form heterodimers in nonallelic combinations. We describe the isolation of a gene, umc1, encoding a MADS-box protein, which displays significant similarity to the Saccharomyces cerevisiae MCM1 gene. umc1 complemented the viability defect of yeast mcm1 mutants. In U. maydis, umc1 deletion mutants were viable and pathogenic development was unaffected. Nevertheless, the basal expression levels of several pheromone-inducible genes were significantly reduced leading to an attenuated mating reaction. In contrast to S. cerevisiae, where Mcm1p plays a crucial role in the cell-type specific expression of a- and α-specific genes, the U. maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes.     

Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step in the cytosolic isoprenoid biosynthesis pathway in higher plants. To understand the contribution of HMGR to plant development, we isolated T-DNA insertion mutants for HMG1 and HMG2. The hmg1 and hmg2 mutants were both more sensitive than the wild type (WT) to lovastatin, an inhibitor of HMGR. The hmg2 mutant showed no visible phenotype under normal growth conditions. In contrast, the hmg1 mutant exhibited dwarfing, early senescence, and sterility. Expression of senescence-associated genes 12 (SAG12), a marker gene for senescence, was induced in the hmg1 mutant at an earlier stage than in the WT. Levels of trans-cytokinins--hormones known to inhibit senescence--were not lower in hmg1. The mutant did not have the typical appearance of brassinosteroid (BR)-deficient mutants, except for a dwarf phenotype, because of the suppression of cell elongation. The expression of several genes involved in cell elongation was suppressed in hmg1. WT plants treated exogenously with inhibitors of sterol biosynthesis had similar gene expression and sterility characteristics as the hmg1 mutants. Pleiotropic phenotypes were rescued by feeding with squalene, the precursor of sterols and triterpenoids. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 plays a critical role in triterpene biosynthesis, and that sterols and/or triterpenoids contribute to cell elongation, senescence, and fertility.     

Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis

It is well established that polarized exocytosis is essential for fungal virulence. By contrast, the contribution of endocytosis is unknown. We made use of a temperature-sensitive mutant in the endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptor Yup1 and demonstrate that endocytosis in Ustilago maydis is essential for the initial steps of pathogenic development, including pheromone perception and cell-cell fusion. Furthermore, spore formation and germination were drastically reduced, whereas colonization of the plant was only slightly inhibited. The function of endocytosis in the recognition of mating pheromone through the G protein-coupled pheromone receptor Pra1 was analyzed in greater detail. Biologically active Pra1-green fluorescent protein localizes to the plasma membrane and is constitutively endocytosed. Yup1(ts) mutants that are blocked in the fusion of endocytic transport vesicles with early endosomes are impaired in pheromone perception and conjugation hyphae formation. This is attributable to an accumulation of Pra1-carrying endocytic vesicles in the cytoplasm and the depletion of the receptor from the membrane. Consistently, strong Pra1 expression rescues the signaling defects in endocytosis mutants, but subsequent cell fusion is still impaired. Thus, we conclude that endocytosis is essential for recognition of the partner at the beginning of the pathogenic program but has additional roles in mating as well as spore formation and germination.     

Role of the Mf1-1 pheromone precursor gene of the filamentous ascomycete Cryphonectria parasitica

Site-directed recombination was used to obtain a Cryphonectria parasitica strain carrying deletions at the Mf1-1 gene locus. Macroscopic features such as growth rate and conidia production were unaffected by Mf1-1 deletions, but, when a strain containing a complete deletion of Mf1-1 was used as spermatia it was male sterile. The same strain was fully competent as a female parent. Deletion of three of the seven putative pheromone peptide repeats within the gene had no effect on mating. Male fertility of the complete deletion strain was restored when an ectopic copy of the Mf1-1 gene was returned by transformation. Expression of the mating type specific pheromone precursor gene Mf1-1 was stimulated by growth in nutritionally poor liquid media. It was found that age and source of inoculum of liquid cultures influences pheromone precusor gene expression, i.e., conidia did not express Mf1-1 and cultures derived from conidia were significantly delayed in expression of this gene, as were cultures derived from young mycelium. Cultures inoculated with older hyphae, however, expressed Mf1-1 within 1 day after inoculation.