The repertoire of effector candidates in Colletotrichum lindemuthianum reveals important information about Colletotrichum genus lifestyle.

The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean (Phaseolus vulgaris), and anthracnose is one of the most devastating diseases of this plant species. However, little is known about the proteins that are essential for the fungus-plant interactions. Knowledge of the fungus' arsenal of effector proteins is of great importance for understanding this pathosystem. In this work, we analyzed for the first time the arsenal of Colletotrichum lindemuthianum effector candidates (ClECs) and compared them with effector proteins from other species of the genus Colletotrichum, providing a valuable resource for studying the infection mechanisms of these pathogens in their hosts. Isolates of two physiological races (83.501 and 89 A2 2-3) of C. lindemuthianum were used to predict 353 and 349 ClECs, respectively. Of these ClECs, 63% were found to be rich in cysteine, have repetitive sequences of amino acids, and/or possess nuclear localization sequences. Several conserved domains were found between the ClECs. We also applied the effector prediction to nine species in the genus Colletotrichum, and the results ranged from 247 predicted effectors in Colletotrichum graminicola to 446 in Colletotrichum orbiculare. Twelve conserved domains were predicted in the effector candidates of all analyzed species of Colletotrichum. An expression analysis of the eight genes encoding the effector candidates in C. lindemuthianum revealed their induction during the biotrophic phase of the fungus on the bean.

The transcriptional landscape of basidiosporogenesis in mature Pisolithus microcarpus basidiocarp.

BACKGROUND: Pisolithus microcarpus (Cooke & Massee) G. Cunn is a gasteromycete that produces closed basidiocarps in symbiosis with eucalypts and acacias. The fungus produces a complex basidiocarp composed of peridioles at different developmental stages and an upper layer of basidiospores free of the hyphae and ready for wind dispersal upon the rupture of the basidiocarp pellis. During basidiosporogenesis, a process that takes place inside the basidiocarp peridioles, a conspicuous reserve of fatty acids is present throughout development. While several previous studies have described basidiosporogenesis inside peridioles, very little is known about gene expression changes that may occur during this part of the fungal life cycle. The objective of this work was to analyze gene transcription during peridiole and basidiospore development, while focusing specifically on cell cycle progression and lipid metabolism. RESULTS: Throughout different developmental stages of the peridioles we analyzed, 737 genes were regulated between adjacent compartments (>5 fold, FDR-corrected p-value < 0.05) corresponding to 3.49% of the genes present in the P. microcarpus genome. We identified three clusters among the regulated genes which showed differential expression between the peridiole developmental stages and the basidiospores. During peridiole development, transcripts for proteins involved in cellular processes, signaling, and information storage were detected, notably those for coding transcription factors, DNA polymerase subunits, DNA repair proteins, and genes involved in chromatin structure. For both internal embedded basidiospores (hereto referred to as "Internal spores", IS) and external free basidiospores (hereto referred to as "Free spores", FS), upregulated transcripts were found to involve primary metabolism, particularly fatty acid metabolism (FA). High expression of transcripts related to ß-oxidation and the glyoxylate shunt indicated that fatty acids served as a major carbon source for basidiosporogenesis. CONCLUSION: Our results show that basidiocarp formation in P. microcarpus involves a complex array of genes that are regulated throughout peridiole development. We identified waves of transcripts with coordinated regulation and identified transcription factors which may play a role in this regulation. This is the first work to describe gene expression patterns during basidiocarp formation in an ectomycorrhizal gasteromycete fungus and sheds light on genes that may play important roles in the developmental process.

Transposable elements belonging to the Tc1-Mariner superfamily are heavily mutated in Colletotrichum graminicola.

Transposable elements are ubiquitous and constitute an important source of genetic variation in addition to generating deleterious mutations. Several filamentous fungi are able to defend against transposable elements using RIP(repeat-induced point mutation)-like mechanisms, which induce mutations in duplicated sequences. The sequenced Colletotrichum graminicola genome and the availability of transposable element databases provide an efficient approach for identifying and characterizing transposable elements in this fungus, which was the subject of this study. We identified 132 full-sized Tc1-Mariner transposable elements in the sequenced C. graminicola genome, which were divided into six families. Several putative transposases that have been found in these elements have conserved DDE motifs, but all are interrupted by stop codons. An in silico analysis showed evidence for RIP-generated mutations. The TCg1 element, which was cloned from the Brazilian 2908 m isolate, has a putative transposase sequence with three characteristic conserved motifs. However, this sequence is interrupted by five stop codons. Genomic DNA from various isolates was analyzed by hybridization with an internal region of TCg1. All of the isolates featured transposable elements that were similar to TCg1, and several hybridization profiles were identified. C. graminicola has many Tc1-Mariner transposable elements that have been degenerated by characteristic RIP mutations. It is unlikely that any of the characterized elements are autonomous in the sequenced isolate. The possible existence of active copies in field isolates from Brazil was shown. The TCg1 element is present in several C. graminicola isolates and is a potentially useful molecular marker for population studies of this phytopathogen.

Boto, a class II transposon in Moniliophthora perniciosa, is the first representative of the PIF/Harbinger superfamily in a phytopathogenic fungus.

Boto, a class II transposable element, was characterized in the Moniliophthora perniciosa genome. The Boto transposase is highly similar to plant PIF-like transposases that belong to the newest class II superfamily known as PIF/Harbinger. Although Boto shares characteristics with PIF-like elements, other characteristics, such as the transposase intron position, the position and direction of the second ORF, and the footprint, indicate that Boto belongs to a novel family of the PIF/Harbinger superfamily. Southern blot analyses detected 6-12 copies of Boto in C-biotype isolates and a ubiquitous presence among the C- and S-biotypes, as well as a separation in the C-biotype isolates from Bahia State in Brazil in at least two genotypic groups, and a new insertion in the genome of a C-biotype isolate maintained in the laboratory for 6 years. In addition to PCR amplification from a specific insertion site, changes in the Boto hybridization profile after the M. perniciosa sexual cycle and detection of Boto transcripts gave further evidence of Boto activity. As an active family in the genome of M. perniciosa, Boto elements may contribute to genetic variability in this homothallic fungus. This is the first report of a PIF/Harbinger transposon in the genome of a phytopathogenic fungus.

The pectate lyase encoded by the pecCl1 gene is an important determinant for the aggressiveness of Colletotrichum lindemuthianum.

Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean, and the genes that encode its cell-wall-degrading enzymes are crucial for the development of the disease. Pectinases are the most important group of cell wall-degrading enzymes produced by phytopathogenic fungi. The pecC1l gene, which encodes a pectate lyase in C. lindemuthianum, was isolated and characterized. Possible cis-regulatory elements and transcription factor binding sites that may be involved in the regulation of genetic expression were detected in the promoter region of the gene. pecCl1 is represented by a single copy in the genome of C. lindemuthianum, though in silico analyses of the genomes of Colletotrichum graminicola and Colletotrichum higginsianum suggest that the genome of C. lindemuthianum includes other genes that encode pectate lyases. Phylogenetic analysis detected two groups that clustered based on different members of the pectate lyase family. Analysis of the differential expression of pecCl1 during different stages of infection showed a significant increase in pecCl1 expression five days after infection, at the onset of the necrotrophic phase. The split-maker technique proved to be an efficient method for inactivation of the pecCl1 gene, which allowed functional study of a mutant with a site-specific integration. Though gene inactivation did not result in complete loss of pectate lyase activity, the symptoms of anthracnose were reduced. Analysis of pectate lyases might not only contribute to the understanding of anthracnose in the common bean but might also lead to the discovery of an additional target for controlling anthracnose.

Efficiency of artificial hybridization in soybean during the summer depending on temperature and relative humidity / Eficiência da hibridação artificial em soja durante o verão depende da temperatura e umidade relativa

Hybridization is an important improvement method used in the soybean culture. However, there is little information on the recommended relative moisture and air temperature degree for artificial pollination. Therefore, this study aimed to determine the efficiency of artificial hybridization between soybean parents according to different periods of the day. Artificial pollination of 14 hybrid combinations occurred in greenhouse in three periods of the day. The parents were: TMG 801, TMG 803, BRSGO 7560, BRS Valiosa RR, Agua-Marinha RR and NK 7059 RR. The studied variables were: relative moisture, air temperature, number of days to flowering, performed artificial pollination, pods without sepal, produced seeds, germinated seeds, hybrid plants and percentage of pods without sepals. Data were submitted to normality and homogeneity of variance test, analysis of variance, Tukey, Scheffé and 2 tests, and correlation analysis. Six hundred and seventy-two artificial pollinations were performed. From which were obtained 436 pods without sepals and approximately 90% of produced seeds was hybrid. The results indicated that artificial pollinations performed in January, with parent used in this study, were more efficient in the period from 10:00 a.m. to 12:00 a.m., with mean relative moisture of 34.1% and mean temperature of 38.5 ºC and 2:00 a.m. to 4:00 p.m. with 30.7% and 41.6 ºC respectively for relative moisture and means of temperature.

A hibridação é um importante método de melhoramento utilizado na cultura da soja. No entanto, são poucas as informações sobre a magnitude da umidade relativa e da temperatura do ar recomendadas para a atividade de polinização artificial. Portanto, objetivou-se determinar a eficiência da hibridação artificial entre genitores de soja em função de diferentes períodos do dia. Efetuaram-se, em casa de vegetação, polinizações artificiais em 14 combinações híbridas em três períodos do dia. Os genitores utilizados foram: TMG 801, TMG 803, BRSGO 7560, BRS Valiosa RR, Água-Marinha RR e NK 7059 RR. As variáveis estudadas foram: umidade relativa, temperatura do ar, número de dias para o florescimento, polinizações artificiais efetuadas, vagens sem sépala, sementes produzidas, sementes germinadas, plantas híbridas e porcentagem de vagens sem sépalas. Os dados foram submetidos ao teste de normalidade e homogeneidade de variância, análise de variância, testes de Tukey, de Scheffé e 2 e análise de correlação. Foram efetuadas 672 polinizações artificiais, nas quais se obtiveram 436 vagens sem sépala. Aproximadamente, 90% das sementes produzidas foram híbridas. Os resultados indicaram que as polinizações artificiais realizadas em janeiro, com os genitores do presente estudo, foram mais eficientes no período das 10:00 h às 12:00 h, com umidade relativa média de 34,1% e temperatura média de 38,5 ºC e das 14:00h às 16:00h com 30,7% e 41,6 ºC, respectivamente para umidade relativa e temperatura média.

Diversidade de características agronômicas e moleculares em cultivares de soja com diferentes graus de resistência à Phakopsora Pachyrhizi / Diversity of agronomic and molecular traits of the soybean cultivars with diferent levels of Phakopsora Pachyrhizi resistance

Com o intuito de orientar melhoristas na escolha de parentais visando à resistência à ferrugem-asiática associada a características de interesse econômico, foram avaliados 17 genótipos de soja quanto às características agronômicas e moleculares por meio de marcadores microssatélites. Foram instalados dois experimentos, o primeiro inoculado com ferrugem-asiática e o segundo sem inoculação. A média de alelos por loco foi igual a 4,56, sendo verificados alelos exclusivos nas cultivares TMG 801, UFUS Impacta, UFUS Xavante, Conquista, BRSGO 7860RR, A 7002 e UFVTN 104. Por meio do índice ponderado, com base no polimorfismo molecular, verificou-se o maior valor de similaridade para as cultivares Conquista e BRS Valiosa RR. O peso médio de 100 sementes foi igual a 14,56g no experimento com ferrugem-asiática, e 18,0g no experimento sem ferrugem-asiática. As médias da proporção de lesões nos folíolos infectados por ferrugem-asiática ocorreram entre 11,48% a 47,18%. Com base nos resultados pôde-se concluir que existe substancial variabilidade genética no germoplasma da soja em relação aos alelos potencialmente ligados aos genes de resistência à ferrugem-asiática. Visando a resistência ou tolerância à ferrugem-asiática associada a características econômicas de interesse seriam mais indicados os cruzamentos das cultivares UFVTN 104 com TMG 801 e/ou UFUS Impacta, Conquista com UFUS Xavante, e UFUS 8011 com BRSGO 7560 e/ou TMG 803.

With intend to direct breeders in choose of parents for Asian soybean rust resistance associated with characteristics of interest economic, agronomic traits and microsatellite markers of 17 genotypes of soybean were evaluated in two experiments, one inoculated with soybean rust and the other without inoculation. The average of alleles per locus was 4.56. The cultivars TMG 801, UFUS Impacta, UFUS Xavante, Conquista, BRSGO 7860RR, A 7002 and UFVTN 104 exhibited exclusive alleles. The results of the weighted índex using molecular markers show the major similarity for the cultivars Conquista and BRS Valiosa RR. The average weight of 100 seeds was equal 14.56g on the experiment with soybean rust and 18.0g on experiment without soybean rust. The average of proportion of infection by soybean rust on the leaf range 11.48% to 47.18%. We conclude that there is substantial genetic variability on soybean germoplasm with relation the allele's potential linked for soybean rust resistance genes. With intend of associate Asian soybean rust resistance with economic interest characteristics will be indicated the mating among UFVTN 104 x TMG 801 and/or UFUS Impacta, Conquista x UFUS Xavante, UFUS 8011 x BRSGO 7560 and/or TMG 803.