Citrus leprosis virus (CiLV), a tentative member of the Rhabdoviridae family, affects citrus trees in Brazil, where it is transmitted by mites Brevipalpus spp. It also occurs in other South American countries and was recently identified in Central America. This northbound spread of CiLV is being considered a serious threat to the citrus industry of the United States. However, despite its importance, difficulties related to the biology of CiLV have hindered much of the progress regarding its accurate detection, leaving both the analyses of symptoms and electron microscopy as the only tools available. An attempt to overcome this problem was made by constructing a cDNA library from double-stranded RNA extracted from leprosis lesions of infected Citrus sinensis (sweet orange) leaves. After cloning and sequencing, specific primers were designed to amplify putative CiLV genome regions with similarity to genes encoding the movement protein and replicase of other plant viruses. RNA from infected citrus plants corresponding to different varieties and locations were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using the two pairs of primers. Amplified products were purified, cloned in pGEM-T, and sequenced. The sequences confirmed the genomic regions previously associated with CiLV. The results demonstrate that RT-PCR was specific, accurate, rapid, and reliable for the detection of CiLV.

The complete nucleotide sequence of the genomic RNA 1 (8745 nt) and RNA 2 (4986 nt) of Citrus leprosis virus cytoplasmic type (CiLV-C) was determined using cloned cDNA. RNA 1 contains two open reading frames (ORFs), which correspond to 286 and 29 kDa proteins. The 286 kDa protein is a polyprotein putatively involved in virus replication, which contains four conserved domains: methyltransferase, protease, helicase and polymerase. RNA 2 contains four ORFs corresponding to 15, 61, 32 and 24 kDa proteins, respectively. The 32 kDa protein is apparently involved in cell-to-cell movement of the virus, but none of the other putative proteins exhibit any conserved domain. The 59 regions of the two genomic RNAs contain a 'cap' structure and poly(A) tails were identified in the 39-terminals. Sequence analyses and searches for structural and non-structural protein similarities revealed conserved domains with members of the genera Furovirus, Bromovirus, Tobravirus and Tobamovirus, although phylogenetic analyses strongly suggest that CiLV-C is a member of a distinct, novel virus genus and family, and definitely demonstrate that it does not belong to the family Rhabdoviridae, as previously proposed. Based on these results it was proposed that Citrus leprosis virus be considered as the type member of a new genus of viruses, Cilevirus.

DNA macroarrays of 279 genes of Xanthomonas axonopodis pv. citri potentially associated with pathogenicity and virulence were used to compare the transcriptional alterations of this bacterium in response to two synthetic media. Data analysis indicated that 31 genes were up-regulated by synthetic medium XVM2, while only 7 genes were repressed. The results suggest that XVM2 could be used as an in vitro system to identify candidate genes involved in pathogenesis of X. axonopodis pv. citri. Within the genus Xanthomonas, several genes have been found associated with pathogenicity and virulence. Of these genes, the avr (avirulence), rpf (named for regulation of pathogenicity factors), and hrp (named for hypersensitive response and pathogenicity) genes are perhaps the most widely studied elements. The avr genes encode a known group of effector proteins responsible for controlling the ability of bacteria to elicit the hypersensitive reaction in resistant hosts (26) and may also participate in pathogenicity or virulence in compatible interactions (38,47). The rpf operon is thought to control the production of important pathogenicity factors, such as proteases, endoglucanases, polygalacturonate ligases, and extracellular polysaccharides (5, 17). Finally, the hrp genes are thought to encode proteins involved in the type III secretion system, responsible for delivering effector proteins inside the host plant cells (8,9,20,21).Even though pathogenicity and virulence of Xanthomonas axonopodis pv. citri have been traditionally associated with the activity of a single avirulence-like gene known as pthA (14,22,45,46), little is known about other gene products involved in these processes. In this respect, transcription profiling under natural conditions may be a good alternative to identify all the elements involved in pathogenicity and virulence of this microbial pathogen. However, leaf spot pathogens, such as X. axonopodis pv. citri, do not reach high population levels in infected tissues and do not yield enough material (either bacterial cells or RNA) to conduct gene expression studies. Therefore, in an attempt to develop an alternative system for gene expression studies, an in vitro system was evaluated in order to determine whether it could be used to model pathogen responses to host tissues under controlled conditions. The two media selected were NB (nutrient broth), commonly used for growing this bacterium, and XVM2, suspected to mimic the environment of the plant intercellular spaces (1, 41, 51, 55).Construction of DNA macroarrays. To study the expression profile of X. axonopodis pv. citri, 279 candidate genes, associated with pathogenicity or virulence by sequence similarity, were selected from the genome of this bacterium (13). Specific primers were designed to amplify either the entire open reading frame or a fragment of it. PCR amplifications were performed in two rounds using 96-well plates and a Mastercycler Eppendorf (Eppendorf). The quality of amplimers was analyzed by agarose gel electrophoresis.Prior to the cons...

The I locus of the common bean, Phaseolus vulgaris, controls the development of four different phenotypes in response to inoculation with Bean common mosaic virus, Bean common mosaic necrosis virus, several other related potyviruses, and one comovirus. We have generated a high-resolution linkage map around this locus and have aligned it with a physical map constructed with BAC clones. These clones were obtained from a library of the cultivar ''Sprite,'' which carries the dominant allele at the I locus. We have identified a large cluster of TIR-NBS-LRR sequences associated within this locus, which extends over a distance .425 kb. Bean cultivars from the Andean or Mesoamerican gene pool that contain the dominant allele share the same haplotypes as revealed by gel blot hybridizations with a TIR probe. In contrast, beans with a recessive allele display simpler and variable haplotypes. A survey of wild accessions from Argentina to Mexico showed that this multigene family has expanded significantly during evolution and domestication. RNA gel blot analysis indicated that the TIR family of genes plays a role in the response to inoculations with BCMV or BCMNV.

Tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria (Xcv) elicits a hypersensitive response (HR) in leaves of Lycopersicon esculentum near-isogenic line (NIL) 216 and pepper genotypes. One cosmid clone (35 kb) selected from a genomic library of a T3 strain induced an HR in all resistant plants. A 1.5-kb active subclone containing the putative avirulence (avr) gene, designated avrXv3, was sequenced. The avrXv3 gene encodes a 654-bp open reading frame (ORF) with no homology to any known gene. Expression studies with a fusion of this gene and uidA indicated that avrXv3 is plant inducible and controlled by the hypersensitivity and pathogenicity (hrp) regulatory system. Mutational analysis and transcription activation assays revealed that AvrXv3 has transcription activation activity in yeast, and that the putative domain responsible for that activity is located at the C terminus of the AvrXv3 protein. Agrobacterium tumefaciens-mediated transient expression confirmed the direct role of AvrXv3 in eliciting the HR in tomato NIL 216 and supported the hypothesis that Avr proteins must be present inside the plant host cell to trigger the HR.

Strains of tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria elicit a hypersensitive response (HR) in leaves of Lycopersicon pennellii LA716. Genetic segregation of the resistance exhibited ratios near 3:1 in F2 populations, which confirmed that a single dominant gene controlled the inheritance of this trait. With the aid of a collection of introgression lines, restriction fragment length polymorphism, and cleaved amplified polymorphic sequence markers, the resistance locus was located on chromosome 3 between TG599 and TG134. An avirulence gene named avrXv4 was also isolated by mobilizing a total of 600 clones from a genomic DNA library of the T3 strain 91-118 into the X. campestris pv. vesicatoria strain ME90, virulent on L. pennellii. One cosmid clone, pXcvT3-60 (29-kb insert), induced HR in resistant plants. The avirulent phenotype of pXcvT3-60 was confirmed by comparing growth rates in planta and electrolyte leakages among transconjugants carrying a mutated or intact clone with the wild-type T3 strain 91-118. A 1.9-kb DNA fragment contained within a 6.8-kb active subclone was sequenced and was determined to carry an open reading frame of 1,077 bp. The predicted AvrXv4 protein exhibits high similarity to members of an emerging new family of bacterial proteins from plant and mammalian pathogens comprising AvrRxv, AvrBsT, YopJ, YopP, AvrA, and YL40.

Nearly 65,000 citrus EST (Expressed Sequence Tags) have been investigated using the CitEST project database. Microsatellites were investigated in the unigene sequences from Citrus spp. and Poncirus trifoliata. From these sequences, approximately 35% of the non-redundant ESTs contained SSRs. The frequencies of different SSR motifs were similar between Citrus spp and trifoliate orange. In general, mononucleotide repeats appeared to be the most abundant SSRs in the CitEST database, but we also identify di-, tri-, tetra-, penta-and hexanucleotide repeats. The AG/CT and AAG/CTT were the most common dinucleotide and trinucleotide motifs, with frequencies of 54.4% and 25.2%, respectively. Primer sequences flanking SSR motifs were successfully designed and synthesized. After in silico polymorphism analysis, a subset of sixty-eight primers was validated in different Citrus spp. and Poncirus trifoliata. PCR-amplification revealed polymorphism in citrus with all tested primer pairs and showed the potential of these markers for linkage mapping. Our study showed that the CitEST database can be exploited for the development of SSR markers that can amplify Citrus spp. and related genus for comparative mapping and other genetic analyses.

The genetic inheritance of resistance to leprosis, the most important viral disease of citrus in Brazil, was characterized through the phenotypic assessment of 143 hybrids resulting from crosses between tangor 'Murcott' (Citrus sinensis x C. reticulata) and sweet orange 'Pêra' (C. sinensis), considered to be resistant and susceptible to the disease, respectively. All plants were grafted onto Rangpur lime (C. limonia) and inoculated with Citrus leprosis virus, cytoplasmic type through the infestation with viruliferous mites, Brevipalpus phoenicis. The experiments were arranged in a completely randomized block design with 10 replicates. Incidence and severity of the disease in leaves and stems as well as plant growth parameters (plant height and stem diameter) were recorded for 3 years after the infestation with the viruliferous mites. The average values of all variables were analyzed using principal component analysis, discriminant factorial analysis, estimation of the clonal repeatability coefficients, and frequency of the distributions of the average values for each measured variable. The principal component analysis resulted in the identification of at least two groups with resistance and susceptibility to leprosis, respectively. About 99% of all hybrids were correctly classified according to the discriminant factorial analysis. The broad-sense heritability coefficients for characteristics associated with incidence and severity of leprosis ranged from 0.88 to 0.96. The data suggest that the inheritance of resistance to leprosis may be controlled by only a few genes.