BackgroundCitrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.ResultsWe have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.ConclusionWe have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

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BackgroundCitrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.ResultsThrough transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands.ConclusionThe identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker.

BackgroundRust caused by Puccinia psidii Winter has been limiting for the establishment of new Eucalyptus plantations, as well as for resprouting of susceptible genetic materials. Identifying host genes involved in defense responses is important to elucidate resistance mechanisms. Reverse transcription-quantitative PCR is the most common method of mRNA quantitation for gene expression analysis. This method generally employs a reference gene as an internal control to normalize results. A good endogenous control transcript shows minimal variation due to experimental conditions.FindingsWe analyzed the expression of 13 genes to identify transcripts with minimal variation in leaves of 60-day-old clonal seedlings of two Eucalyptus clones (rust-resistant and susceptible) subjected to biotic (P. psidii) and abiotic (acibenzolar-S-methyl, ASM) stresses.ConclusionsFor tissue samples of clones that did not receive any stimulus, a combination of the eEF2 and EglDH genes was the best control for normalization. When pathogen-inoculated and uninoculated plant samples were compared, eEF2 and UBQ together were more appropriate as normalizers. In ASM-treated and untreated leaves of both clones, transcripts of the CYP and elF4B genes combined were the ones with minimal variation. Finally, when comparing expression in both clones for ASM-treated leaves, P. psidii-inoculated leaves, ASM-treated plus P. psidii-inoculated leaves, and their respective controls, the genes with the most stable expression were EgIDH and UBQ. The chitinase gene, which is highly expressed in studies on plant resistance to phytopathogens, was used to confirm variation in gene expression due to the treatments.

To identify genes that are up and down-regulated by water deficit in sugarcane we used the macroarray methodology and the expression level of 3 575 independent sugarcane cDNAs was measured by hybridization with RNA extracted from plants submitted to mild, moderate and severe water deficit. We identified approximately 1 670 differentially expressed genes from which 62 % were up-regulated by different stress-conditions, whereas many repressed genes were exclusive for each time-point. Analysis of similarity showed that approximately 24 % of the differentially expressed genes shared homology with proteins involved in different processes such as signal transduction, hormone metabolism, photosynthesis, transcription and stress response. Transcripts with no known function accounted for approximately 39 % and those without similarity represented 36 % of the sequences. Five genes analyzed by RT-PCR confirmed the macroarray results.

Xanthomonas citri subsp. citri (Xcc) causes citrus canker, a worldwide disease found mainly in sweet oranges (Citrus sinensis (L.) Osbeck). The expression of nine candidate internal reference genes was analyzed in Xcc grown alone and during C. sinensis infection to identify genes most suitable for comparative expression studies in Xcc using reverse transcription quantitative PCR (qRT-PCR). The stability of these genes was determined using the programs geNorm, NormFinder and BestKeeper. The genes most suitable for data normalization during C. sinensis infection were atpD, rpoB, gyrA and gyrB. The use of at least three reference genes is essential for accurate data normalization in Xcc.

Citrus canker is a major disease affecting citrus production in Brazil. It’s mainly caused by Xanthomonas citri subsp. citri strain 306 pathotype A (Xac). We analysed the differential expression of proteins secreted by wild type Xac and an asymptomatic mutant for hrpB4 (ΔhrpB4) grown in Nutrient Broth (NB) and a medium mimicking growth conditions in the plant (XAM1). This allowed the identification of 55 secreted proteins, of which 37 were secreted by both strains when cultured in XAM1. In this secreted protein repertoire, the following stand out: Virk, Polyphosphate-selective porin, Cellulase, Endoglucanase, Histone-like protein, Ribosomal proteins, five hypothetical proteins expressed only in the wild type strain, Lytic murein transglycosylase, Lipoprotein, Leucyl-tRNA synthetase, Co-chaperonin, Toluene tolerance, C-type cytochrome biogenesis membrane protein, Aminopeptidase and two hypothetical proteins expressed only in the ΔhrpB4 mutant. Furthermore, Peptidoglycan-associated outer membrane protein, Regulator of pathogenicity factor, Outer membrane proteins, Endopolygalacturonase, Chorismate mutase, Peptidyl-prolyl cis-trans isomerase and seven hypothetical proteins were detected in both strains, suggesting that there was no relationship with the secretion mediated by the type III secretory system, which is not functional in the mutant strain. Also worth mentioning is the Elongation factor Tu (EF-Tu), expressed only the wild type strain, and Type IV pilus assembly protein, Flagellin (FliC) and Flagellar hook-associated protein, identified in the wild-type strain secretome when grown only in NB. Noteworthy, that FliC, EF-Tu are classically characterized as PAMPs (Pathogen-associated molecular patterns), responsible for a PAMP-triggered immunity response. Therefore, our results highlight proteins potentially involved with the virulence. Overall, we conclude that the use of secretome data is a valuable approach that may bring more knowledge of the biology of this important plant pathogen, which ultimately can lead to the establishment of new strategies to combat citrus canker.

ResumoO presente trabalho teve como objetivo avaliar o crescimento inicial, a massa específica básica, a produção da biomassa e analisar quimicamente a madeira e casca de dois clones de Eucalyptus (um híbrido espontâneo de Eucalyptus urophylla S.T. Blake; um híbrido tri-cross de Eucalyptus urophylla S.T. Blake x (E. camaldulensis Dehn x E. grandis Hill ex Maiden)) e um híbrido de Corymbia citriodoraA. Johnson, distribuídos em três espaçamentos de plantio: 3x3 m; 3x1,5 m; e 3x1 m, na idade de 12 meses. O estudo foi conduzido num experimento localizado no município de Itamarandiba, MG, em delineamento experimental inteiramente casualizado com quatro repetições, em um esquema fatorial 3x3, considerando como níveis dos fatores os diferentes clones e os espaçamentos de plantio. Observou-se para os três clones, que o espaçamento 3x3 m proporcionou plantas com maior crescimento em diâmetro à altura do peito (DAP) em relação aos demais espaçamentos. Os diferentes espaçamentos de plantio não induziram a variação da massa específica básica da madeira e das cascas nos clones estudados. O aumento da densidade de plantio apresentou relação direta com a produção de biomassa por unidade de área. Observou-se decréscimo dos teores de lignina na madeira, para os dois clones de Eucalyptus, do maior espaçamento para os espaçamentos mais adensados. Palavras-chave: Floresta energética; Biomassa; Biocombustível. Abstract Evaluation Corymbia and Eucalyptus clones under different spacings order to produce bioenergy.This study aimed to evaluate the initial growth, the basic density, the production of biomass and chemically analyzing of the wood and bark of two Eucalyptus clones (a hybrid of Eucalyptus urophylla S.T. Blake, a hybrid tri-cross Eucalyptus urophylla S.T. Blake x (E. camaldulensis Dehn x E. grandis Hill ex Maiden)) and a hybridJohnson distributed in three planting spacings: 3x3m, 3x1,5m, 3x1m, at the age of 12 months. The study was conducted in an experiment located in the county Itamarandiba, MG, in a completely randomized design with four replications, in a factorial 3x3, considering how levels of the factors the different clones and planting spacings. It was observed for the three clones that the 3x3m spacing provided plants with greater growth in diameter at breast height (DAP) in relation to the other spacings. The different planting spacings did not induce the variation of the basic specific mass of the wood and the bark in the studied clones. The increase in planting density had a direct relation with biomass production per unit area. There was a decrease in lignin contents in the wood for two clones of Eucalyptus, the larger spacing for the denser spacings. Keywords: Energy forest; Biomass; Biofuel. INTRODUÇÃOA forte pressão exercida pela sociedade para a redução da emissão de poluentes impõe a uma maior busca por biocombustíveis provenientes de fontes renováveis, e nesse cenário, destaca-se a biomassa florestal (CARNEIRO et al., 2014).As espécies do gênero Eucalyptus e Corymbia se destacam entre as diversas opções pa...