Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil

Visando esclarecer alguns eventos bioquímicos durante o processo de infecção no patossistema Puccinia psidii x eucalipto, comparou-se o metabolismo relacionado à defesa em folhas em diferentes níveis de desenvolvimento, em clones (Eucalyptus grandis x E. urophylla) denominados VR e C0, com sessenta dias de idade, resistente e suscetível à ferrugem, respectivamente, determinando-se a atividade de peroxidases e quitinases. Cada tratamento foi composto de 4 repetições, em delineamento inteiramente casualizado, considerando: 2 clones, inoculados e não inoculados com P. psidii; pulverizados com acibenzolar-S-metil (ASM) e água destilada; representados pelo 1º, 2º e 4º pares de folha (com tamanho equivalente a 1/5, 2/5 e 4/5 do desenvolvimento foliar total, respectivamente). A coleta das folhas ocorreu em 4 períodos: 0, 24, 72 e 96 horas após a inoculação (HAI). Os resultados obtidos evidenciaram que apesar de apresentarem níveis superiores de atividade de quitinases e peroxidases, o efeito do ASM ou o efeito da P. psidii não alteraram a atividade dessas enzimas em folhas desenvolvidas (4º par) no decorrer do período. Alterações nos níveis das enzimas após a inoculação apenas foram observadas nas folhas em desenvolvimento (1º e 2º pares), evidenciando que a resposta à infecção foi acompanhada pela síntese dessas enzimas. Os maiores incrementos nas atividades enzimáticas ocorreram nas plantas do clone resistente 72 HAI e nas plantas do clone suscetível que foram tratadas previamente com ASM e posteriormente inoculadas com o P. psidii.To elucidate some biochemical processes during infection in the pathosystem Puccinia psidii x eucalyptus, the defense metabolism in different-stage leaves was compared between rust-resistant and susceptible clones, respectively. In addition, chitinase and peroxidase activities were assayed. Each treatment consisted of 4 replicates, in a completely randomized design: 2 clones, inoculated and not inoculated with P. psidii; sprayed with acibenzolar-S-methyl (ASM) and distilled water; and represented by the 1st leaf pair (size equivalent to 1/5 total leaf development), 2nd pair (2/5 total development), and 4th pair (4/5 total leaf length). Leaves were harvested in 4 periods: 0, 24, 72 and 96 hours after inoculation. Results indicated that ASM treatment or P. psidii action led to higher chitinase and peroxidase activity level but did not alter the expression of these activities in developed leaves (4th pair) during the experiment. Alterations in enzyme levels after inoculation were only observed in developing leaves (1st and 2nd pairs), which suggests that the response to infection was concomitant to chitinase and peroxidase synthesis. The highest increases in enzymatic activities were observed in resistant clones at 72 hours after inoculation and in susceptible ones previously treated with ASM and later inoculated with the pathogen.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Global gene expression of Poncirus trifoliata, Citrus sunki and their hybrids under infection of Phytophthora parasitica

<p>Abstract</p> <p>Background</p> <p>Gummosis and root rot caused by <it>Phytophthora </it>are among the most economically important diseases in citrus. Four F₁resistant hybrids (Pool R), and four F₁susceptible hybrids (Pool S) to <it>P. parasitica</it>, were selected from a cross between susceptible <it>Citrus sunki </it>and resistant <it>Poncirus trifoliata </it>cv. Rubidoux. We investigated gene expression in pools of four resistant and four susceptible hybrids in comparison with their parents 48 hours after <it>P. parasitica </it>inoculation. We proposed that genes differentially expressed between resistant and susceptible parents and between their resistant and susceptible hybrids provide promising candidates for identifying transcripts involved in disease resistance. A microarray containing 62,876 UniGene transcripts selected from the CitEST database and prepared by NimbleGen Systems was used for analyzing global gene expression 48 hours after infection with <it>P. parasitica</it>.</p> <p>Results</p> <p>Three pairs of data comparisons (<it>P. trifoliata</it>/<it>C. sunki</it>, Pool R/<it>C. sunki </it>and Pool R/Pool S) were performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 3.0, 21 UniGene transcripts common to the three pairwise comparative were found to be up-regulated, and 3 UniGene transcripts were down-regulated. Among them, our results indicated that the selected transcripts were probably involved in the whole process of plant defense responses to pathogen attack, including transcriptional regulation, signaling, activation of defense genes participating in HR, single dominant genes (<it>R gene</it>) such as TIR-NBS-LRR and RPS4 and switch of defense-related metabolism pathway. Differentially expressed genes were validated by RT-qPCR in susceptible and resistant plants and between inoculated and uninoculated control plants</p> <p>Conclusions</p> <p>Twenty four UniGene transcripts were identified as candidate genes for <it>Citrus </it>response to <it>P. parasitica</it>. UniGene transcripts were likely to be involved in disease resistance, such as genes potentially involved in secondary metabolite synthesis, intracellular osmotic adjustment, signal transduction pathways of cell death, oxidative burst and defense gene expression. Furthermore, our microarray data suggest another type of resistance in <it>Citrus</it>-<it>Phytophthora </it>interaction conferred by single dominant genes (<it>R gene</it>) since we encountered two previously reported <it>R genes </it>(TIR-NBS-LRR and RPS4) upregulated in the resistant genotypes relative to susceptible. We identified 7 transcripts with homology in other plants but yet unclear functional characterization which are an interesting pool for further analyses and 3 transcripts where no significant similarity was found. This is the first microarray study addressing an evaluation of transcriptional changes in response to <it>P. parasitica </it>in <it>Citrus</it>.</p

Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR

<p>Abstract</p> <p>Background</p> <p>Rust caused by <it>Puccinia psidii </it>Winter has been limiting for the establishment of new <it>Eucalyptus </it>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.</p> <p>Findings</p> <p>We analyzed the expression of 13 genes to identify transcripts with minimal variation in leaves of 60-day-old clonal seedlings of two <it>Eucalyptus </it>clones (rust-resistant and susceptible) subjected to biotic (<it>P. psidii</it>) and abiotic (acibenzolar-S-methyl, ASM) stresses.</p> <p>Conclusions</p> <p>For tissue samples of clones that did not receive any stimulus, a combination of the <it>eEF2 </it>and <it>EglDH </it>genes was the best control for normalization. When pathogen-inoculated and uninoculated plant samples were compared, <it>eEF2 </it>and <it>UBQ </it>together were more appropriate as normalizers. In ASM-treated and untreated leaves of both clones, transcripts of the <it>CYP </it>and <it>elF4B </it>genes combined were the ones with minimal variation. Finally, when comparing expression in both clones for ASM-treated leaves, <it>P. psidii</it>-inoculated leaves, ASM-treated plus <it>P. psidii</it>-inoculated leaves, and their respective controls, the genes with the most stable expression were <it>EgIDH </it>and <it>UBQ</it>. 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.</p

Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress

Avaliação de métodos de inoculação de Phytophthora parasitica em plântulas e plantas jovens de citros

Este trabalho teve como objetivo avaliar métodos de inoculação de Phytophthoraparasitica em plântulas e plantas jovens de citros (Citrus spp.) visando sua utilização em estudos de resistência de porta-enxertos à gomose de Phytophthora. Os métodos de inoculação testados foram: contato planta sem ferimento-patógeno, casca destacada, inserção de disco de meio de cultura contendo micélio sob a casca, método do disco e inserção de agulha e palito infestados em plântulas e plantas jovens de citros. A resistência de genótipos à gomose pode ser avaliada em plântulas in vitro, através de inserção de agulha infestada. O método do disco e o da inserção sob casca foram os melhores quando utilizados em plantas jovens. A medida da área total da lesão é a variável ideal para avaliação da doença. No entanto, o comprimento da lesão pode ser utilizado na avaliação da doença em plântulas, plantas jovens e no campo.The present work was aimed at evaluating inoculation methods of Phytophthoraparasitica on citrus (Citrus spp.) seedlings and young plants to determine the resistance of citrus rootstocks to Phytophthora gummosis. The following inoculation methods were tested: non-wounded host-pathogen contact, bark removal, insertion of culture disc under bark, insertion of inoculated needle or toothpick in the collar region and, the culture disc method. The assessment of resistance to gummosis in citrus seedlings produced in vitro was achieved by inserting an inoculatum in collar region. The disc method and insertion of disc under the bark were the best inoculation methods for young plants under both orchard and greenhouse conditions. The best variable for disease assessment had been the measurement of the total lesion area. Lesion length can also be used when carried out in nurseries and in the field

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil To elucidate some biochemical processes during infection in the pathosystem Puccinia psidii x eucalyptus, the defense metabolism in different-stage leaves was compared between rust-resistant and susceptible clones, respectively. In addition, chitinase and peroxidase activities were assayed. Each treatment consisted of 4 replicates, in a completely randomized design: 2 clones, inoculated and not inoculated with P. psidii; sprayed with acibenzolar-S-methyl (ASM) and distilled water; and represented by the 1(st) leaf pair (size equivalent to 1/5 total leaf development), 2(nd) pair (2/5 total development), and 4(th) pair (4/5 total leaf length). Leaves were harvested in 4 periods: 0, 24, 72 and 96 hours after inoculation. Results indicated that ASM treatment or P. psidii action led to higher chitinase and peroxidase activity level but did not alter the expression of these activities in developed leaves (4(th) pair) during the experiment. Alterations in enzyme levels after inoculation were only observed in developing leaves (1(st) and 2(nd) pairs), which suggests that the response to infection was concomitant to chitinase and peroxidase synthesis. The highest increases in enzymatic activities were observed in resistant clones at 72 hours after inoculation and in susceptible ones previously treated with ASM and later inoculated with the pathogen