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.

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.

Huanglongbing (HLB) is a destructive disease of citrus caused by phloem-limited bacteria, namely 'Candidatus Liberibacter asiaticus' (Las), 'Candidatus Liberibacter africanus', and 'Candidatus Liberibacter americanus'. Although there are no known HLB-resistant citrus species, studies have reported Poncirus trifoliata as being more tolerant. Assuming that callose deposition in the phloem of infected plants can inhibit translocation of photosynthetic products and cause starch accumulation, we compared callose deposition in petioles and starch accumulation in infected leaves of three genotypes (Citrus sinensis, C. sunki, and P. trifoliata) and 15 hybrids (C. sunki × P. trifoliata). Compared with the mock-inoculated plants, higher bacterial counts and greater accumulation of callose and starch were found in C. sinensis, C. sunki, and 10 of the hybrid plants. Lower titer and fewer metabolic changes due to Las infection were observed in P. trifoliata and in two Las-positive hybrids while three hybrids were Las-negative. Callose accumulation was linked to and correlated with genes involved in phloem functionality and starch accumulation was linked to up-regulation of genes involved in starch biosynthesis and repression of those related to starch breakdown. Lower expression of genes involved in phloem functionality in resistant and tolerant plants can partially explain the absence of distinct disease symptoms associated with starch accumulation that are usually observed in HLB-susceptible genotypes.

Huanglongbing (HLB), caused by the bacterium 'Candidatus Liberibacter' spp., is currently one of the most serious diseases of citrus plants and has caused substantial economic losses. Thus far, there is no source of genetic resistance to HLB in the genus Citrus or its relatives. However, several studies have reported Poncirus trifoliata and some of its hybrids to be more tolerant to the disease. The main objective of this study was to report differences in the incidence of 'Ca. L. asiaticus' infection in citrandarin plants, hybrids from Sunki mandarin (Citrus sunki (Hayata) hort. ex Tanaka), and trifoliate orange Rubidoux (P. trifoliata (L.) Raf.)), after conducting an extensive survey under field conditions. These hybrid plants were established for approximately 7 years in an area with a high incidence of 'Ca. L. asiaticus'-infected plants. We selected two experimental areas (area A and area B), located approximately 10 m apart. Area A consists of Pera sweet orange (C. sinensis (L.) Osb.) grafted onto 56 different citrandarin rootstocks. Area B consists of citrandarin scions grafted onto Rangpur lime (C. limonia Osb.) rootstock. Bacteria in the leaves and roots were detected using real-time quantitative polymerase chain reaction. The incidence of 'Ca. L. asiaticus'-infected plants was 92% in area A and 14% in area B. Because infected plants occurred in both areas, we examined whether the P. trifoliata hybrid rootstock influenced HLB development and also determined the distribution of 'Ca. L. asiaticus' in Citrus tree tissues. Although this survey does not present evidence regarding the resistance of P. trifoliata and its hybrids in relation to bacteria or psyllids, future investigation, mainly using the most promising hybrids for response to 'Ca. L. asiaticus', will help us to understand the probable mechanism of defense or identifying compounds in P. trifoliata and its hybrids that are very important as strategy to combat HLB. Details of these results are presented and discussed in this article.

BackgroundGummosis and root rot caused by Phytophthora are among the most economically important diseases in citrus. Four F1 resistant hybrids (Pool R), and four F1 susceptible hybrids (Pool S) to P. parasitica, were selected from a cross between susceptible Citrus sunki and resistant Poncirus trifoliata cv. Rubidoux. We investigated gene expression in pools of four resistant and four susceptible hybrids in comparison with their parents 48 hours after P. parasitica 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 P. parasitica.ResultsThree pairs of data comparisons (P. trifoliata/C. sunki, Pool R/C. sunki 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 (R gene) 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 plantsConclusionsTwenty four UniGene transcripts were identified as candidate genes for Citrus response to P. parasitica. 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 Citrus-Phytophthora interaction conferred by single dominant genes (R gene) since we encountered two previously reported R genes (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 P. parasitica in Citrus.

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.

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