Weighted gene coexpression correlation network analysis reveals the potential molecular regulatory mechanism of citrate and anthocyanin accumulation between postharvest 'Bingtangcheng' and 'Tarocco' blood orange fruit.

BACKGROUND: Organic acids and anthocyanins are the most important compounds for the flavor and nutritional quality of citrus fruit. However, there are few reports on the involvement of co-regulation of citrate and anthocyanin metabolism. Here, we performed a comparative transcriptome analysis to elucidate the genes and pathways involved in both citrate and anthocyanin accumulation in postharvest citrus fruit with 'Tarocco' blood orange (TBO; high accumulation) and 'Bingtangcheng' sweet orange (BTSO; low accumulation). RESULTS: A robust core set of 825 DEGs were found to be temporally associated with citrate and anthocyanin accumulation throughout the storage period through transcriptome analysis. Further according to the results of weighted gene coexpression correlation network analysis (WGCNA), the turquoise and brown module was highly positively correlated with both of the content of citrate and anthocyanin, and p-type ATPase (PH8), phosphoenolpyruvate carboxylase kinase (PEPCK), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H) and glutathione S transferase (GST) were considered key structural genes. Moreover, MYB family transcription factor (PH4), Zinc finger PHD-type transcription factor (CHR4, HAC12), Zinc finger SWIM-type transcription factor (FAR1) and Zinc finger C3H1-type transcription factor (ATC3H64) were considered hub genes related to these structural genes. Further qRT-PCR analysis verified that these transcription factors were highly expressed in TBO fruit and their expression profiles were significantly positively correlated with the structural genes of citrate and anthocyanin metabolism as well as the content of citrate and anthocyanin content. CONCLUSIONS: The findings suggest that the CHR4, FAR1, ATC3H64 and HAC12 may be the new transcription regulators participate in controlling the level of citrate and anthocyanin in postharvest TBO fruit in addition to PH4. These results may providing new insight into the regulation mechanism of citrate and anthocyanin accumulation in citrus fruit.

Metastable nano-zirconium phosphate inside gel-type ion exchanger for enhanced removal of heavy metals.

Nanotechnology has provided a new opportunity for water decontamination from trace heavy metals, yet the relatively poor acidic stability remains a major obstacle for the nano-adsorbents, given that acidic treatment is frequently used to regenerate the heavy metal-saturated adsorbents. Zirconium phosphate (ZrP) is very promising for water treatment due to its absolute insoluble nature, though it interacts with heavy metals mainly through the non-specific electrostatic attraction. Herein, we prepared the ultrafine ZrP (~3.9 nm) inside the commercially available gel-type cation exchanger (N001), i.e., the sulfonated poly(styrene-co-divinylbenzene) bead. The resultant nanocomposite ZrP@N001 contained the amorphous nanoparticles (NPs) with metastable γ-ZrP structure as the main phase, unlike the layered α-ZrP formed inside the macroporous cation exchanger D001 (referred to as ZrP@D001). As a result, ZrP@N001 could selectively adsorb heavy metals through inner-sphere coordination, possessing a much stronger adsorption affinity than ZrP@D001, as confirmed by XPS analysis. In both batch and column assays on the Pb(II)-polluted water, ZrP@N001 exhibited superior adsorption performance over ZrP@D001. After adsorption, the exhausted ZrP@N001 was fully refreshed by acidic treatment for a 5-cyclic adsorption-regeneration run with constant removal efficiencies. This study may open a door for the rational design of highly efficient water purifiers for heavy metal control.

Biotechnological Approaches for Genetic Improvement of Lemon (Citrus limon (L.) Burm. f.) against Mal Secco Disease.

Among Citrus species, lemon is one of the most susceptible to mal secco disease, a tracheomycosis caused by the mitosporic fungus Plenodomus tracheiphilus, which induces chlorosis followed by leaf drop and progressive desiccation of twigs and branches. Severe infection can cause the death of the plant. Since no effective control strategies are available to efficiently control the pathogen spread, host tolerance is the most desirable goal in the struggle against mal secco disease. To date, both traditional breeding programs and biotechnological techniques were not efficient in developing novel varieties coupling tolerance to mal secco with optimal fruit quality. Furthermore, the genetic basis of host resistance has not been fully deciphered yet, hampering the set-up of marker-assisted selection (MAS) schemes. This paper provides an overview of the biotechnological approaches adopted so far for the selection of mal secco tolerant lemon varieties and emphasizes the promising contribution of marker-trait association analysis techniques for both unraveling the genetic determinism of the resistance to mal secco and detecting molecular markers that can be readily used for MAS. Such an approach has already proved its efficiency in several crops and could represent a valuable tool to select novel lemon varieties coupling superior fruit quality traits and resistance to mal secco.

Enhanced removal of arsenic from water by using sub-10 nm hydrated zirconium oxides confined inside gel-type anion exchanger.

Given high selectivity and excellent stability, zirconium oxides are very promising in selective removal of arsenic, fluorine, and phosphorus from water. Nevertheless, it remains challenging to prepare sub-10 nm zirconium oxides of ultra-high adsorptive reactivity. Herein, we prepared hydrated zirconium oxides (HZO) of 4.88 ± 1.02 nm by conducting in-situ precipitation of nanoparticles (NPs) inside the gel-type anion exchanger (GAE). GAE was swollen in water and contained lots of < 10 nm swollen pores, restricting excess growth of HZO NPs. In comparison, the NPs formed inside the macroporous anion exchanger (MAE) possessed an average diameter of 30.91 ± 8.98 nm. XPS O1s analysis indicated that the oxygen sites in the gel-type nanocomposite (HZO@GAE) possessed a much higher proportion (48.9%) of reactive terminal oxygen (-OH) than the macroporous nanocomposite (HZO@MAE, 21.2%). Thus, HZO@GAE exhibited significantly enhanced adsorption reactivity toward As(V)/As(III) than HZO@MAE. The exhausted HZO@GAE could be fully regenerated by alkali treatment for repeated use without any loss in decontamination efficiency. In column assays, the HZO@GAE column successively produced ~2400 bed volume (BV) clean water ([As]<10 µg/L) from synthetic groundwater, exceeding twice the amount produced by the HZO@MAE column. This study may shed new light on developing highly efficient nanocomposites for water decontamination.

Characterization of the binding ability of the odorant binding protein BminOBP9 of Bactrocera minax to citrus volatiles.

BACKGROUND: Bactrocera minax, one of the most important citrus pests, oviposits exclusively on citrus fruit. In the insect olfactory system, odorant-binding proteins (OBPs) facilitate the initial recognition role of host odor molecules. The aim of this study was to characterize the functional OBPs of B. minax and identify specific volatile organic compounds in the Citrus genus as OBP targets. RESULTS: BminOBP9 (BminGOBP99a), a closely related homolog of BdorGOBP99a, which reduces the egg-laying behavior of Bactrocera dorsalis through silencing technology, was cloned, expressed, and purified. The binding ability of BminOBP9 to 11 citrus volatiles was then examined using fluorescence competition binding assays (FCBA). The results demonstrated that BminOBP9 could bind to all tested citrus volatiles, as could BdorGOBP99a, ZcucGOBP99a, and ZtauGOBP99a. Interestingly, the binding ability of BminOBP9 was the strongest among the four, suggesting that BminOBP9 may have a function in the specific recognition of citrus volatiles. Furthermore, we aligned the above four proteins and found nine distinctive amino acid sites in BminOBP9. To identify the unique binding sites of BminOBP9, we produced the nine mutants using site-directed mutagenesis. Further FCBA showed that the binding ability of the nine mutants to citrus volatiles significantly reduced, and six of them (substitutes S24P, L36F, E53K, N68D, D112A, and S118R) had the weakest binding ability. CONCLUSION: The results demonstrated that BminOBP9 was the specific protein involved in the perception of citrus host volatiles by B. minax. Moreover, BminOBP9 could prove efficient in screening the candidate odors for pest management. © 2020 Society of Chemical Industry.

Citron C-05 inhibits both the penetration and colonization of Xanthomonas citri subsp. citri to achieve resistance to citrus canker disease.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a serious bacterial disease that affects citrus production worldwide. Citron C-05 (Citrus medica) is the only germplasm in the Citrus genus that has been identified to exhibit strong resistance to Xcc. However, it has not been determined when, where, and how Xcc is restricted in the tissues of Citron C-05 during the infection process. In the present study, we investigated the spatiotemporal growth dynamics of an eGFP-labeled virulent Xcc (eGFP-Xcc) strain in Citron C-05 along with five susceptible biotypes (i.e., lemon, pummelo, sour orange, sweet orange, and ponkan mandarin) upon inoculation via the spraying or leaf infiltration of a bacterial suspension. The results from extensive confocal laser scanning microscopy analyses showed that while Xcc grew rapidly in plants of all five susceptible genotypes, Xcc was severely restricted in the epidermal and mesophyll cell layers of the leaves of Citron C-05 in the early stage of infection. Not surprisingly, resistance against Xcc in Citron C-05 was found to be associated with the production of reactive oxygen species and hypersensitive response-like cell death, as well as greater upregulation of several defense-related genes, including a pathogenesis-related gene (PR1) and a glutathione S-transferase gene (GST1), compared with sweet orange as a susceptible control. Taken together, our results not only provide further valuable details of the spatiotemporal dynamics of the host entry, propagation, and spread of Xcc in both resistant and susceptible citrus plants but also suggest that resistance to Xcc in Citron C-05 may be attributed to the activation of multiple defense mechanisms.

Substantial Equivalence of a Transgenic Lemon Fruit Showing Postharvest Fungal Pathogens Resistance.

The development of genetically modified (GM) crops speeds up the obtainment of novel varieties with improved agronomic characteristics. However, the risk evaluation of the use of GMs is mandatory before their release in the market. In this paper, an untargeted and comprehensive nuclear magnetic resonance-based metabolomic study was carried out on the peel and flesh of a transgenic lemon clone (E23) expressing the chit42 gene and exhibiting an increased tolerance to some pathogenic fungi and on its wild type. Results highlighted a substantial equivalence of the metabolomics profile of the transgenic clone compared to the wild type. In addition, an enhanced response of the E23 clone toward fungal pathogens affecting the postharvest management in lemon was evidenced. These results confirm the potential of genetic engineering for the punctual modification of specific agronomic traits without altering the whole pattern of metabolites and open new perspectives for a more sustainable and effective management of specific postharvest diseases in citrus.

A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants.

Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase (PDS) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.

Endogenous auxin and its manipulation influence in vitro shoot organogenesis of citrus epicotyl explants.

Endogenous auxin is an important regulator of in vivo organ development, but its role in in vitro organogenesis is unclear. It has been observed that the basal end of epicotyl cuttings of juvenile citrus seedlings produces fewer shoots than the apical end. Here, we report that elevated endogenous auxin levels in the basal end of citrus epicotyl cuttings are inhibitory for in vitro shoot organogenesis. Using transgenic citrus plants expressing an auxin-inducible GUS reporter gene, we have observed elevated levels of auxin at the basal end of stem cuttings that are mediated by polar auxin transport. Depleting endogenous auxin or blocking polar auxin transport enhances shoot organogenesis. An auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), can also enhance shoot organogenesis independent of its action on polar auxin transport. Finally, we demonstrate that the promotional effects of depleting endogenous auxin or blocking polar auxin transport on shoot organogenesis are cytokinin-dependent. Our study thus provides meaningful insights into possible roles of endogenous auxin and polar auxin transport, as well as auxin-cytokinin interactions, in in vitro shoot organogenesis. Meanwhile, our results may also provide practical strategies for improving in vitro shoot organogenesis for citrus and many other plant species.

Elevated auxin and reduced cytokinin contents in rootstocks improve their performance and grafting success.

Plant grafting is an important technique for horticultural and silvicultural production. However, many rootstock plants suffer from undesirable lateral bud outgrowth, low grafting success rates or poor rooting. Here, we used a root-predominant gene promoter (SbUGT) to drive the expression of a tryptophan-2-monooxygenase gene (iaaM) from Agrobacterium tumefaciens to increase auxin levels in tobacco. The transgenic plants, when used as a rootstock, displayed inhibited lateral bud outgrowth, enhanced grafting success rate and improved root initiation. However, root elongation and biomass of SbUGT::iaaM transgenic plants were reduced compared to those of wild-type plants. In contrast, when we used this same promoter to drive CKX (a cytokinin degradation gene) expression, the transgenic tobacco plants displayed enhanced root elongation and biomass. We then made crosses between the SbUGT::CKX and SbUGT::iaaM transgenic plants. We observed that overexpression of the CKX gene neutralized the negative effects of auxin overproduction on root elongation. Also, the simultaneous expression of both the iaaM and CKX genes in rootstock did not disrupt normal growth and developmental patterns in wild-type scions. Our results demonstrate that expression of both the iaaM and CKX genes predominantly in roots of rootstock inhibits lateral bud release from rootstock, improves grafting success rates and enhances root initiation and biomass.

Population structure and diversity of citrus tristeza virus (CTV) isolates in Hunan province, China.

Stem-pitting (SP) is the main type of citrus tristeza virus (CTV) that causes severe damage to citrus trees, especially those of sweet orange, in Hunan province, China. Understanding the local CTV population structure should provide clues for effective mild strain cross-protection (MSCP) of the SP strain of CTV. In this study, markers for the p23 gene, multiple molecular markers (MMMs), and sequence analysis of the three silencing suppressor genes (p20, p23 and p25) were employed to analyze the genetic diversity and genotype composition of the CTV population based on 51 CTV-positive samples collected from 14 citrus orchards scattered around six major citrus-growing areas of Hunan. The results indicated that the CTV population structure was extremely complex and that infection was highly mixed. In total, p23 gene markers resulted in six profiles, and MMMs demonstrated 25 profiles. The severe VT and T3 types appeared to be predominantly associated with SP, while the mild T30 and RB types were related to asymptomatic samples. Based on phylogenetic analysis of the amino acid sequences of p20, p23 and p25, 19 representative CTV samples were classified into seven recently established CTV groups and a potentially novel one. A high level of genetic diversity, as well as potential recombination, was revealed among different CTV isolates. Five pure SP severe and two pure mild strains were identified by genotype composition analysis. Taken together, the results update the genetic diversity of CTV in Hunan with the detection of one possible novel strain, and this information might be applicable for the selection of appropriate mild CTV strains for controlling citrus SP disease through cross-protection.

Dual and Opposing Roles of Xanthine Dehydrogenase in Defense-Associated Reactive Oxygen Species Metabolism in Arabidopsis.

While plants produce reactive oxygen species (ROS) for stress signaling and pathogen defense, they need to remove excessive ROS induced during stress responses in order to minimize oxidative damage. How can plants fine-tune this balance and meet such conflicting needs? Here, we show that XANTHINE DEHYDROGENASE1 (XDH1) in Arabidopsis thaliana appears to play spatially opposite roles to serve this purpose. Through a large-scale genetic screen, we identified three missense mutations in XDH1 that impair XDH1's enzymatic functions and consequently affect the powdery mildew resistance mediated by RESISTANCE TO POWDERY MILDEW8 (RPW8) in epidermal cells and formation of xanthine-enriched autofluorescent objects in mesophyll cells. Further analyses revealed that in leaf epidermal cells, XDH1 likely functions as an oxidase, along with the NADPH oxidases RbohD and RbohF, to generate superoxide, which is dismutated into H2O2 The resulting enrichment of H2O2 in the fungal haustorial complex within infected epidermal cells helps to constrain the haustorium, thereby contributing to RPW8-dependent and RPW8-independent powdery mildew resistance. By contrast, in leaf mesophyll cells, XDH1 carries out xanthine dehydrogenase activity to produce uric acid in local and systemic tissues to scavenge H2O2 from stressed chloroplasts, thereby protecting plants from stress-induced oxidative damage. Thus, XDH1 plays spatially specified dual and opposing roles in modulation of ROS metabolism during defense responses in Arabidopsis.

The relationship between PthA expression and the pathogenicity of Xanthomonas axonopodis pv. citri.

Citrus canker disease, caused by Xanthomonas axonopodis pv. citri, affects almost all citrus species and cultivars and hascaused severe damage to the citrus industry worldwide. PthA is considered the main pathogenesis effector of the pathogen. This research aimed to temporally and spatially analyze the expression of the PthA protein of the bactrium during its culture, and then try to understand the relationship between the PthA expression levels and the pathogenicity. The relationship between the expression of PthA and the pathogenicity of X. axonopodis pv. citri was fully investigated by using SDS-PAGE, Western blot, ELISA and field inoculation, It was found that bacteria cultured for 36 h had the highest expression of PthA and showed the most virulent pathogenicity. The conservation duration of the pathogen isolates influenced their PthA expression and the pathogenicity, and negative relationship between the duration and the expression of PthA and pathogenicity. When the stored pathogen bacteria were cultured in liquid LB medium, they were able to regain activated, showing higher PthA expression level and enhanced pathogenicity, even though the activity was inferior, in terms of both PthA expression and pathogenicity, than the freshly isolated ones. Seven isolates from different citrus orchards displayed almost identical protein expression profiles. It could conclude that the expressions of PthA was positively related to pathogenicity.

A universal oligonucleotide microarray with a minimal number of probes for the detection and identification of viroids at the genus level.

A major challenge in the agricultural industry is the development of techniques that can screen plant samples for viroid infection. Microarrays are promising in this regard, as their high throughput nature can potentially allow for the detection of a range of viroids in a single test. In this paper we present a microarray that can detect a wide spectrum of all 8 reported viroid genera including 37 known plant viroid species. The array was constructed using an automated probe design protocol which generated a minimal number of probes to detect viroids at the genus level. The designed microarray showed a high specificity and sensitivity when tested with a set of standard virus samples. Finally, the microarray was applied to screen infected field samples, with Hop stunt viroid infection identified as the major disease causing pathogen for an infected citrus sample.

An improvement of shotgun proteomics analysis by adding next-generation sequencing transcriptome data in orange.

BACKGROUND: Shotgun proteomics data analysis usually relies on database search. Because commonly employed protein sequence databases of most species do not contain sufficient protein information, the application of shotgun proteomics to the research of protein sequence profile remains a big challenge, especially to the species whose genome has not been sequenced yet. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we present a workflow with integrated database to partly address this problem. First, we downloaded the homologous species database. Next, we identified the transcriptome of the sample, created a protein sequence database based on the transcriptome data, and integtrated it with homologous species database. Lastly, we developed a workflow for identifying peptides simultaneously from shotgun proteomics data. CONCLUSIONS/SIGNIFICANCE: We used datasets from orange leaves samples to demonstrate our workflow. The results showed that the integrated database had great advantage on orange shotgun proteomics data analysis compared to the homologous species database, an 18.5% increase in number of proteins identification.

Construction of EGFP-labeling system for visualizing the infection process of Xanthomonas axonopodis pv. citri in planta.

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker, an economically important disease to world citrus industry. To monitor the infection process of Xac in different citrus plants, the enhanced green florescent protein (EGFP) visualizing system was constructed to visualize the propagation and localization in planta. First, the wild-type Xac was isolated from the diseased leaves of susceptible 'Bingtang' sweet orange, and then the isolated Xac was labeled with EGFP by triparental mating. After PCR identification, the growth kinetics and pathogenicity of the transformants were analyzed in comparison with the wild-type Xac. The EGFP-labeled bacteria were inoculated by spraying on the surface and infiltration in the mesophyll of 'Bingtang' sweet orange leaves. The bacterial cell multiplication and diffusion processes were observed directly under confocal laser scanning microscope at different intervals after inoculation. The results indicated that the EGFP-labeled Xac releasing clear green fluorescence light under fluorescent microscope showed the infection process and had the same pathogenicity as the wild type to citrus. Consequently, the labeled Xac demonstrated the ability as an efficient tool to monitor the pathogen infection.

Ptcorp gene induced by cold stress was identified by proteomic analysis in leaves of Poncirus trifoliata (L.) Raf.

A proteomic approach was employed to investigate the cold stress-responsive proteins in trifoliate orange (Poncirus trifoliata (L.) Raf.), which is a well-known cold tolerant citrus relative and widely used as rootstock in China. Two-year-old potted seedlings were exposed to freezing temperature (-6°C) for 50 min (nonlethal) and 80 min (lethal), and the total proteins were isolated from leaves of the treated plants. Nine differentially accumulated proteins over 2-fold changes in abundance were identified by two-dimensional gel electrophoresis and mass spectrometry. Among these proteins, a resistance protein induced by the nonlethal cold treatment (protein spot #2 from P. trifoliata) was selected as target sequence for degenerated primer design. By using the designed primers, a PCR product of about 700 bp size was amplified from P. trifoliata genomic DNA, which was further cloned and sequenced. A nucleotide sequence of 676 bp was obtained and named Ptcorp. Blast retrieval showed that Ptcorp shared 88% homology with an EST of cold acclimated Bluecrop (Vaccinium corymbosum) library (Accession number: CF811080), indicating that Ptcorp had association with cold acclimation. Semiquantitative RT-PCR analysis demonstrated that Ptcorp gene was up-regulated by cold stress which was consistent with the former result of protein expression profile. As the resistance protein (NBS-LRR disease resistance protein family) gene was up-regulated by cold stress in trifoliate orange and satsuma mandarin, it may imply that NBS-LRR genes might be associated with cold resistance in citrus.

Construction of tandem repeats of DNA fragments by a polymerase chain reaction-based method.

We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71 bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.

Selection of reference genes for quantitative real-time RT-PCR analysis in citrus.

Quantitative real-time reverse transcription polymerase chain reaction (qPCR) has become the preferred method for studying low-abundant mRNA expression. Appropriate application of qPCR in such studies requires the use of reference gene(s) as an internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. Expression of the reference gene should be independent from development stage, cell/tissue types, treatments and environmental conditions. Recognizing the importance of reference gene(s) in normalization of qPCR data, various reference genes have been evaluated for stable expression under specific conditions in various organisms. In plants, only a few of them have been investigated, and very few reports about such reference genes in citrus. In the present study, seven candidate reference genes (18SrRNA, ACTB, rpII, UBQI, UBQ10, GAPDH and TUB) were tested, and three of them (18SrRNA, ACTB and rpII) proved to be the most stable ones among six leaf samples of different citrus genotypes. The three candidate reference genes were further analyzed for their stability of expression in five different tissues, and the results indicated that they were not completely stable. It is commonly accepted that gene expression studies should be normalized using more than one reference gene. Based on our results, we propose the use of the mean result rendered by18SrRNA, ACTB and rpII as reference genes to normalize mRNA levels in qPCR analysis of diverse cultivars and tissues of citrus. These results may provide a guideline for future works on gene expression in citrus by using qPCR.

Transformation of sweet orange [Citrus sinensis (L.) Osbeck] with pthA-nls for acquiring resistance to citrus canker disease.

The COOH terminal of pthA encoding three nuclear localizing signals (NLS) was amplified by polymerase chain reaction (PCR) from the plasmid of Xanthomonas axonopodis pv. citri, the pathogen of citrus canker disease. Then the sense and antisense strands of the nls were cloned into pBI121 vector. pthA-nls driven by the CaMV35 s promoter was transferred into sweet orange via Agrobacterium -mediated transformation. Successful integration was confirmed by PCR and Southern blotting, and 12 sense-nls (nls (+)) and 9 antisense-nls (nls (-)) transgenic clones were obtained. The expression of nls fragment was analyzed by RT-PCR, Real time q-PCR and Western blotting, in which the specific NLS protein was detected only in nls (+) transgenic clones. In an in vitro assay, when pin-puncture inoculation was performed with 2.5 × 10(7) cfu/ml of bacterial solution, the nls (+) transgenic clones showed no typical lesion development, while typical symptoms were observed in the wild types and the nls (-) transgenic clones. In vivo assay results indicated that the nls (+) transgenic clones showed less disease incidence, in comparison with the wild types and the nls (-) transgenic clones, when pin-puncture inoculation was performed with 10(4)-10(5) cfu/ml. The minimum disease incidence was 23.3% for 'Sucarri' sweet orange and 33.3% for 'Bingtang' sweet orange. When 10(4)-10(7) cfu/ml of pathogen was spray inoculated, the nls (+) transgenic clones did not show any symptom, and even the concentration raised to 10(9) cfu/ml, the disease incidence was 20-80%, while the wild types and the nls (-) transgenic clones had 100% disease development with whatever concentration of inoculum. Two transgenic clones were confirmed to be resistant to citrus canker disease in the repeated inoculation. The results suggested that the transformation of nls sense strands may offer an effective way to acquire resistance to citrus canker disease.