Acquisition and transmission of selected CTV isolates by Aphis gossypii

Citrus tristeza virus (CTV) is a severe threat to the citrus industry. Disease symptoms and severity may vary depending on the CTV isolates. These are responsible for the decline of trees grafted on sour orange rootstock, or stem pitting on some citrus commercial cultivars regardless of rootstock. In the Calabria region (Italy), CTV was first reported on cultivars imported from other countries. However, recent observations suggested that natural spread of CTV was occurring and a study was needed to determine the epidemiological status and aphid transmission of CTV in Calabria. The role played by local A. gossypii in the spread of CTV was analyzed in the laboratory using various viral acquisition, inoculation periods with three different CTV isolates. Single aphid vectors acquired CTV after a minimum of 30 min acquisition access period (AAP) and were able to transmit the virus after a 60 min inoculation access period (IAP) to healthy plants. A minimum of four aphid vectors were needed to reach 50% transmission probability. The results suggested that the three tested strains are transmitted by A. gossypii in a semi-persistent mode. The results demonstrated that local A. gossypii population can acquire and transmit efficiently the tested virus isolates with serious implications on the virus spread.     

Citrus tristeza virus replicates and forms infectious virions in protoplasts of resistant citrus relatives

Citrus tristeza virus (CTV) is the most economically important viral disease of citrus worldwide. Cultivars with improved CTV tolerance or resistance are needed to manage CTV-induced diseases. The citrus relatives Poncirus trifoliata (L.) Raf., Swinglea glutinosa (Blanco) Merr., and Severinia buxifolia (Poir) Ten. are potential sources of CTV resistance, but their resistance mechanisms are poorly characterized. As a first step to examine the mechanisms of resistance to CTV in these citrus relatives and selected Citrus × Poncirus hybrids, it was necessary to develop methods for protoplast isolation and viral inoculation to allow examination of CTV multiplication in this range of citrus varieties and relatives. Leaf and/or cultured cell protoplasts were isolated and inoculated with four biologically distinct CTV isolates. Northern-blot hybridization analyses for progeny RNAs and immuno-electron microscopy assays for newly produced virions showed that CTV replicated and produced infectious particles in protoplasts from all of the resistant plants tested. These results suggest that resistance to CTV observed at the plant level results from a lack of virus movement and/or some induced resistance response, rather than lack of viral multiplication at the cellular level.     

Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem‐associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23‐kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N‐terminal 157‐amino‐acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV‐like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23‐derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem‐specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV‐specific symptoms (vein clearing and necrosis, and stem pitting), but not the non‐specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem‐specific accumulation of the p23Δ158–209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N‐terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.     

Spatial and temporal spread of Citrus tristeza virus and its aphid vectors in the North western area of Morocco

First report of Citrus tristeza virus (CTV,Closterovirus) in Morocco datesback to 1961 in collections of citrus varieties. An exhaustive survey of citrus in the north of the country in 2009 revealed that CTV was spread all over the citrus production area. We attempted to evaluate the relative contribution of different aphid species in the spread of CTV disease in a Citrus reticulata orchard at the Loukkous region during 2 years (2012 and 2013). The overall CTV incidence estimated in the experimental site increased from 17.8% in 2012 to 31.15% in 2013. The most abundant aphid species colonising clementine trees was Aphis spiraecola and A. gossypii. Both aphid species reached their maximum peaks during the spring season. The rate of viruliferous aphids, estimated by real‐time RT‐PCR of single aphid, revealed that 35.4% of winged A. gossypii and 28.8% of winged A. spiraecola were viruliferous, confirming a high inoculum pressure in the area surrounding the experimental site. The aphid species Toxoptera citricida, which is able to transmit the aggressive isolates of CTV, was not found in the Loukkous region. The study of the spatial distribution of the CTV showed that in general, the disease was randomly distributed in the field. Overall, the results seem to indicate that A. spiraecola may be considered as the major aphid species contributing to CTV spread in our experimental conditions. The prevalence of mild strains in the region and the high level of aphid flight activity could explain the rapid evolution of CTV incidence in the experimental area.     

Citrus tristeza virus infection induces the accumulation of viral small RNAs (21–24-nt) mapping preferentially at the 3′-terminal region of the genomic RNA and affects the host small RNA profile

To get an insight into the host RNA silencing defense induced by Citrus tristeza virus (CTV) and into the counter defensive reaction mediated by its three silencing suppressors (p25, p20 and p23), we have examined by deep sequencing (Solexa-Illumina) the small RNAs (sRNAs) in three virus-host combinations. Our data show that CTV sRNAs: (i) represent more than 50% of the total sRNAs in Mexican lime and sweet orange (where CTV reaches relatively high titers), but only 3.5% in sour orange (where the CTV titer is significantly lower), (ii) are predominantly of 21–22-nt, with a biased distribution of their 5′ nucleotide and with those of (+) polarity accumulating in a moderate excess, and (iii) derive from essentially all the CTV genome (ca. 20 kb), as revealed by its complete reconstruction from viral sRNA contigs, but adopt an asymmetric distribution with a prominent hotspot covering approximately the 3′-terminal 2,500 nt. These results suggest that the citrus homologues of Dicer-like (DCL) 4 and 2 most likely mediate the genesis of the 21 and 22 nt CTV sRNAs, respectively, and show that both ribonucleases act not only on the genomic RNA but also on the 3′ co-terminal subgenomic RNAs and, particularly, on their double-stranded forms. The plant sRNA profile, very similar and dominated by the 24-nt sRNAs in the three mock-inoculated controls, was minimally affected by CTV infection in sour orange, but exhibited a significant reduction of the 24-nt sRNAs in Mexican lime and sweet orange. We have also identified novel citrus miRNAs and determined how CTV influences their accumulation.     

Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing

RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.     

A Turnip crinkle virus Isolate Lacking the CP Counter‐Defence Protein Gene Providing Protection Against the Wild‐Type Strain is Associated with Highly Localized RNA Silencing

Cross‐protection has been used successfully and commercially to control a range of virus diseases for which the selection of suitable mild strains of plant viruses is necessary. Turnip crinkle virus (TCV) is highly pathogenic on Arabidopsis plants and its silencing suppressor‐defective mutant, TCVΔCP, can induce highly localized RNA silencing which is differs from that of other protective strains. We found that TCVΔCP provides some protection against wild‐type TCV but lacks complete protection, and the relative locations of the protective virus and challenge virus affect the degree of cross‐protection. However, similar cross‐protection afforded by TCVΔCP is not observed in Nicotiana benthamiana plants. As expected, TCVΔCP pre‐infected Arabidopsis plants fail to protect against infection with the unrelated Cucumber mosaic virus, strain Fhy. It appears that cross‐protection afforded by TCVΔCP requires that the challenge virus be very similar in sequence, which is a characteristic of RNA silencing. In order to investigate whether the protection is associated with the highly localized RNA silencing, mutant plants involved in key silencing pathway genes of RNA silencing machinery, including dcl2, dcl4 and triple dcl2/dcl3/dcl4 mutants were used. The results demonstrate that cross‐protection afforded by TCVΔCP is dependent on host RNA silencing, and both DCL2 and DCL4 play important roles in this process.     

Overview of Strain Characterization in Relation to Serological and Molecular Detection of Citrus tristeza <i>Closterovirus</i>

Tristeza is a devastating viral disease in all the citrus growing countries throughout the world and has killed millions of citrus trees in severely affected orchards. The citrus species grafted on sour orange rootstock are affected by this disease. Predominantly, the sweet orange, grapefruit and lime trees grafted on sour orange exhibit severe symptoms like quick decline, vein clearing, pin holing, bark scaling and degeneration leading to variable symptoms. Symptomatic expression of Citrus tristeza virus (CTV) in different hosts has been attributed to virus isolates which are from severe to mild. Different serological and molecular assays have been deployed to differentiate the strains of CTV. Citrus tristeza virus is diversified towards its strains on the basis of biological, serological and molecular characterization. Phenotypic expression is due to genetic alteration and different molecular basis have now been adopted for strain differentiation. This review will give a brief idea about the different CTV isolates, their characterization based on nucleic acid and serological assays. Different methods along with salient features for strain characterization has also been reviewed. This review will also open the new aspects towards formulation of management strategies through different detection techniques.     

Pathological categorization of the stem‐pitting and mild strains Citrus tristeza virus in Taiwan and their genomic analysis

Citrus tristeza virus (CTV), the causal agent of tristeza disease, causes the devastating diseases worldwide. In Taiwan, complex cultivars and long‐term infection by CTV result in more than 90% of infected citrus trees, but local strain identification and classification are still incomplete. Here, six CTV strains were categorized by grafting onto eight citrus cultivars and the pathological characteristics of the stem‐pitting mild strains were identified. After 6 months of inoculation, the pummelo stem‐pitting severe strain (CTV‐Pum/SP/T1) only caused severe symptoms in Wentan pummelo (WP) and the mild strain (CTV‐Pum/M/T5) was symptomless in every cultivar; the sweet orange (SO) stem‐pitting severe strain (CTV‐SwO/SP/T7) affected SO, WP and Ponkan mandarin (PM), and the mild strain (CTV‐SwO/M/T51) caused no symptoms in SO except for WP; the mandarin stem‐pitting severe strain (CTV‐Man/SP/T46) caused severe impacts in PM, WP and Eureka lemon, whereas the mild strain (CTV‐Man/M/T2) only caused severe stem‐pitting in WP. The full‐length sequencing of both pummelo stem‐pitting strains and phylogenetic analysis revealed that CTV‐Pum/SP/T1 and CTV‐Pum/M/T5 were related to the HA18‐9 and HA16‐5 strains from Hawaii, respectively. Moreover, recombination analysis revealed that TCT repeat sequences existed at open reading frame 1a in both the CTV‐Pum/SP/T1 and the T36 strains from the United States, indicating that the possible evolution relationship between two regions. Furthermore, improved universal and specific primer pairs were designed for more specific, sensitive detection to meet the needs for quarantine and early prevention. The understanding of strain pathogenicity and genomic analysis provided further characterization of each strain and enabled practical challenge inoculation against CTV disease.     

Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding

The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus‐based vector (clbvINpr‐AtFT and clbvINpr‐CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4–6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr‐AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.     

Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus

Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded.     

Structure‐function analysis of npr1 alleles in Arabidopsis reveals a role for its paralogs in the perception of salicylic acid

Salicylic acid (SA) is necessary for plant defence against some pathogens, whereas NPR1 is necessary for SA perception. Plant defence can be induced to an extreme by several applications of benzothiadiazole (BTH), an analogue of SA. Thus, plants that do not perceive BTH grow unaffected, whereas wild‐type plants grow stunted. This feature allows us to screen for mutants in Arabidopsis thaliana that show insensitivity to BTH in a high‐throughput fashion. Most of the mutants are npr1 alleles, with similar phenotypes in plant weight and pathogen growth. The mutations are clustered in the carboxyl‐terminal part of the protein, and no obvious null alleles were recovered. These facts have prompted a search for knockouts in the NPR1 gene. Two of these KO alleles identified are null and have an intermediate phenotype. All the evidence presented lead us to propose a redundancy in SA perception, with the paralogs of NPR1 taking part in this signalling. We show that the mutations recovered in the screening genetically interact with the paralogs preventing their function in SA signalling.     

Sensitive and Specific Digoxigenin-labelled RNA Probes for Routine Detection of Citrus tristeza virus by Dot-blot Hybridization

A non‐radioactive dot‐blot hybridization assay for the successful detection of Citrus tristeza virus (CTV) RNA in total nucleic acid extracts of infected citrus was developed. Two digoxigenin (DIG)‐labelled minus‐sense riboprobes, complementary to the coat protein gene sequence of a Chinese and an Apulian CTV isolate were synthesized. Several citrus tissues were evaluated as optimal virus source and leaf petioles were found appropriate material for reliable detection. The hybridization assay showed a detection limit corresponding to 0.2 mg of fresh infected tissue. The riboprobes allowed CTV detection in isolates from different geographical areas, grown in the screenhouse or in the field, resulting in similar hybridization patterns. The infected trees were tested during different seasons with positive results, although from July to August most of the samples gave a weaker hybridization signal, compared to other seasons. The high sensitivity and reliability of the molecular hybridization assay described make it a good alternative to serological methods for CTV detection.     

Transgenic expression in citrus of single-chain antibody fragments specific to <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> confers virus resistance

Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40–60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.