The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes

Background Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. Results In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. Conclusions NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the “arms race” with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes

Background Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. Results In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. Conclusions NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the “arms race” with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

Ferrochelatase is a conserved downstream target of the blue light-sensing White collar complex in fungi.

Light is a universal signal perceived by organisms, including fungi, in which light regulates common and unique biological processes depending on the species. Previous research has established that conserved proteins, originally called White collar 1 and 2 from the ascomycete Neurospora crassa, regulate UV/blue light sensing. Homologous proteins function in distant relatives of N. crassa, including the basidiomycetes and zygomycetes, which diverged as long as a billion years ago. Here we conducted microarray experiments on the basidiomycete fungus Cryptococcus neoformans to identify light-regulated genes. Surprisingly, only a single gene was induced by light above the commonly used twofold threshold. This gene, HEM15, is predicted to encode a ferrochelatase that catalyses the final step in haem biosynthesis from highly photoreactive porphyrins. The C. neoformans gene complements a Saccharomyces cerevisiae hem15Delta strain and is essential for viability, and the Hem15 protein localizes to mitochondria, three lines of evidence that the gene encodes ferrochelatase. Regulation of HEM15 by light suggests a mechanism by which bwc1/bwc2 mutants are photosensitive and exhibit reduced virulence. We show that ferrochelatase is also light-regulated in a white collar-dependent fashion in N. crassa and the zygomycete Phycomyces blakesleeanus, indicating that ferrochelatase is an ancient target of photoregulation in the fungal kingdom.

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe.

A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe.

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex.

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.

Interaction of Cryptococcus neoformans Rim101 and protein kinase A regulates capsule.

Cryptococcus neoformans is a prevalent human fungal pathogen that must survive within various tissues in order to establish a human infection. We have identified the C. neoformans Rim101 transcription factor, a highly conserved pH-response regulator in many fungal species. The rim101 multiply sign in circle mutant strain displays growth defects similar to other fungal species in the presence of alkaline pH, increased salt concentrations, and iron limitation. However, the rim101 multiply sign in circle strain is also characterized by a striking defect in capsule, an important virulence-associated phenotype. This capsular defect is likely due to alterations in polysaccharide attachment to the cell surface, not in polysaccharide biosynthesis. In contrast to many other C. neoformans capsule-defective strains, the rim101 multiply sign in circle mutant is hypervirulent in animal models of cryptococcosis. Whereas Rim101 activation in other fungal species occurs through the conserved Rim pathway, we demonstrate that C. neoformans Rim101 is also activated by the cAMP/PKA pathway. We report here that C. neoformans uses PKA and the Rim pathway to regulate the localization, activation, and processing of the Rim101 transcription factor. We also demonstrate specific host-relevant activating conditions for Rim101 cleavage, showing that C. neoformans has co-opted conserved signaling pathways to respond to the specific niche within the infected host. These results establish a novel mechanism for Rim101 activation and the integration of two conserved signaling cascades in response to host environmental conditions.

A PCR-based method to characterise and identify benzimidazole resistance in Helminthosporium solani

Control of Helminthosporium solani, the cause of silver scurf in potato tubers, has been impaired by selection of benzimidazole-resistant strains as a result of repeated use of the fungicide thiabendazole. Identification of thiabendazole-resistant strains of H. solani by conventional techniques takes several weeks. Primers designed from conserved regions of the fungal beta-tubulin gene were used to PCR amplify and sequence a portion of the gene. A point mutation was detected at codon 198 in thiabendazole-resistant isolates causing a change in the amino acid sequence from glutamic acid to alanine or glutamine. Species-specific PCR primers designed to amplify this region were used in conjunction with a restriction endonuclease to cause cleavage in sensitive isolates only and thus provide a rapid diagnostic test to differentiate field isolates.

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and tile wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total B-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, ± 10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mM in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict Properly up to 50-fold higher hexokinase activity.

COX24 codes for a mitochondrial protein required for processing of the COX1 transcript

In most strains of Saccharomyces cerevisiae the mitochondrial gene COX1, for subunit 1 of cytochrome oxidase, contains multiple exons and introns. Processing of COX1 primary transcript requires accessory proteins factors, some of which are encoded by nuclear genes and others by reading frames residing in some of the introns of the COX1 and COB genes. Here we show that the low molecular weight protein product of open reading frame YLR204W, for which we propose the name COX24, is also involved in processing of COX1 RNA intermediates. The growth defect of cox24 mutants is partially rescued in strains harboring mitochondrial DNA lacking introns. Northern blot analyses of mitochondrial transcripts indicate cox24 null mutants to be blocked in processing of introns aI2 and aI3. The dependence of intron aI3 excision on Cox24p is also supported by the growth properties of the cox24 mutant harboring mitochondrial DNA with different intron compositions. The intermediate phenotype of the cox24 mutant in the background of intronless mitochondrial DNA, however, suggests that in addition to its role in splicing of the COX1 pre-mRNA, Cox24p still has another function. Based on the analysis of a cox14-cox24 double mutant, we propose that the other function of Cox24p is related to translation of the COX1 mRNA. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Translational regulation of nuclear gene COX4 expression by mitochondrial content of phosphatidylglycerol and cardiolipin in Saccharomyces cerevisiae.

Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Delta) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5' and 3' untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of a functional PGS1 gene under control of the ADH1 promoter restored phosphatidylglycerol synthesis and expression of mtGFP. Deletion analysis of the 5' UTR(COX4) revealed the presence of a 50-nucleotide fragment with two stem-loops as a cis-element inhibiting COX4 translation. Binding of a protein factor(s) specifically to this sequence was observed with cytoplasm from pgs1Delta but not PGS1 cells. Using HIS3 and lacZ as reporters, extragenic spontaneous recessive mutations that allowed expression of His3p and beta-galactosidase were isolated, which appeared to be loss-of-function mutations, suggesting that the genes mutated may encode the trans factors that bind to the cis element in pgs1Delta cells.

The role of Sse1 in the de novo formation and variant determination of the [PSI+] prion.

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion "strains" or variants--a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes--continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating [PSI+] de novo formation and variant determination. Overproduction of the Sse1 chaperone dramatically enhanced [PSI+] formation whereas deletion of SSE1 severely inhibited it. Only an unstable weak [PSI+] variant was formed in SSE1 disrupted cells whereas [PSI+] variants ranging from very strong to very weak were formed in isogenic wild-type cells under identical conditions. Thus, Sse1 is essential for the generation of multiple [PSI+] variants. Mutational analysis further demonstrated that the physical association of Sse1 with Hsp70 but not the ATP hydrolysis activity of Sse1 is required for the formation of multiple [PSI+] variants. Our findings establish a novel role for Sse1 in [PSI+] de novo formation and variant determination, implying that the mammalian Hsp110 may likewise be involved in the etiology of protein-folding diseases.

Differential gene expression by Paracoccidioides brasiliensis in host interaction conditions: Representational difference analysis identifies candidate genes associated with fungal pathogenesis

Paracoccidioides brasiliensis causes infection by the host inhalation of airborne propagules of the mycelia phase of the fungus. These particles reach the lungs, and disseminate to virtually all organs. Here we describe the identification of differentially expressed genes in studies of host-fungus interaction. We analyzed two cDNA populations of P. brasiliensis, one obtained from infected animals and the other an admixture of fungus and human blood thus mimicking the hematologic events of the fungal dissemination. Our analysis identified transcripts differentially expressed. Genes related to iron acquisition, melanin synthesis and cell defense were specially upregulated in the mouse model of infection. The upregulated transcripts of yeast cells during incubation with human blood were those predominantly related to cell wall remodeling/synthesis. The expression pattern of genes was independently confirmed in host conditions, revealing their potential role in the infection process. This work can facilitate functional studies of novel regulated genes that may be important for the survival and growth strategies of P. brasiliensis in humans. (c) 2006 Elsevier Masson SAS. All rights reserved.

Induction, expression and characterisation of laccase genes from the marine-derived fungal strains Nigrospora sp. CBMAI 1328 and Arthopyrenia sp. CBMAI 1330

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)