Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

Inheritance of resistance to Plum pox virus in apricot (Prunus armeniaca L.)

The inheritance of resistance to Plum pox virus (PPV) has been studied in 1,178 apricot hybrids. Seven hundred and eighteen F1 hybrids, obtained from controlled crosses between the susceptible Greek cultivar “Bebecou” and the resistant PPV cultivars of American origin (“Stark Early Orange,” ‘NJA2,” ‘Veecot,” “Sunglo,” “Harlayne,” and “Orangered”) were evaluated for resistance to the PPV-M (Marcus) strain, 8 years after artificial inoculation. The inheritance of resistance to PPV has been additionally studied for the first time in a BC₁ population of 95 apricot hybrids for four vegetative periods. Reaction of each hybrid to PPV-M was scored through visual symptoms, indexing onto GF-305 and double-antibody sandwich enzyme-linked immunosorbent assay tests. Segregation within the hybrids, determined by Chi-squared analysis, fits a 1:1 ratio (P ≤ 0.05) of the resistant vs susceptible, indicating that resistance to PPV is controlled by a single dominant gene locus and that the above six resistant cultivars are heterozygous for the trait. Plants carrying this gene may initially develop disease symptoms on leaves but eventually recover and no virus can be detected in leaves. Susceptible plants show similar symptoms initially but remain symptomatic. Inheritance of resistance to PPV also has been studied in 365 F1 hybrids by crossing the resistant cultivar “Stella” with the susceptible “Bebecou” and the resistant cultivars “Sunglo” and “NJA2,” for 8 years after inoculation. The segregation ratio was 1:0 (resistant/susceptible) suggesting that “Stella” is homozygous for the resistance trait. The purpose of this work was the enhancement of the knowledge of inheritance of resistance to PPV for the selection of new cultivars.     

Isozyme profiles associated with the hypersensitive response of Chenopodium foetidum to plum pox virus infection

Isozyme profiles of esterases (E.C. 3.1.1.1), glutamate oxaloacetate transaminase (E.C. 2.6.1.1) and peroxidases (E.C. 1.11.1.7) have been determined in healthy tissues of Chenopodium foetidum as well as their modifications during leaf development. The effect of plum pox virus infection on the isozyme profiles has also been studied. Virus-induced necrotic lesions displayed a peroxidase (POX) pattern that has not been found in any other tissue of the plant so far analyzed. The pattern was similar to that of old yellow leaves, except that POX-B, which was detected in the necrotic lesions, has not been detected in any developmental stage of healthy leaves. Changes in the peroxidase profile seem to begin early during infection, even before necrosis is visible. We suggest that senescence is established at necrotic lesions extending from there to the rest of the infected leaf affecting the peroxidase isozyme pattern. However, other changes, which induce POX-B, must also take place at necrotic lesions. These do not extend to the rest of the infected leaves. Plum pox virus infection has less effect on the glutamate oxaloacetate transaminase and esterase isozyme patterns, inducing an almost normal senescence pattern.     

Localisation of plum pox virus in apricot stem and petiole tissues by tissue printing onto nitrocellulose membrane

The localisation of plum pox virus (PPV) in stem and petiole tissues of nine susceptible apricot cultivars and GF305 peach seedling has been studied. From stem and petioles consecutive transverse sections spaced at 1 mm were made and tissue sections printed onto nitrocellulose membrane. The resulting prints were probed with a specific antibody for plum pox virus, followed by a rabbit anti-goat antibody conjugated with horse radish peroxidase, in order to localise the virus within the tissues. In stems the virus was mainly present in xylem and pith. The possible presence of the virus in the sclerenchyma is discussed. In petioles the virus was present in epidermis and parenchymas, but not in vessels. The probable movement through the xylem and from cell to cell has been shown.     

Generation and characterisation of functional recombinant antibody fragments against RNA replicase NIb from plum pox virus

A monoclonal antibody (mAb 2A) able to react against the RNA replicase NIb from plum pox virus (PPV) was obtained and used for generating a specific scFv fragment. The VH and VL coding sequences were cloned and expressed as a fusion scFv protein to alkaline phosphatase. This fusion protein was able to recognise viral NIb in both Western and tissue-print ELISA blots. The affinity and specificity of scFv2A for NIb was similar to that of the parental mAb and the region YLEAFY from PPV-NIb was identified by PEPSCAN assay as the putative epitope. Isolated VH domains from scFv2A were also expressed as fusion to alkaline phosphatase. However, their ability to react against NIb was greatly altered. scFv2A fragments were transiently expressed in the cytosol of Nicotiana benthamiana and although they accumulated to low levels, inhibition-ELISA results indicated that they retained antigen-binding activity.     

Signaling of systemic acquired resistance in tobacco depends on ethylene perception

The hypersensitive interaction between Tobacco mosaic virus (TMV) and tobacco results in accumulation of salicylic acid (SA), defense gene expression, and development of systemic acquired resistance (SAR) in uninfected leaves. The plant hormones SA and ethylene have been implicated in SAR. From a study with ethylene-insensitive (Tetr) tobacco, we concluded that ethylene perception is required to generate the systemic signal molecules in TMV-infected leaves that trigger SA accumulation, defense gene expression, and SAR development in uninfected leaves. Ethylene perception was not required for the responses of the plant to the systemic signal that leads to SAR development.     

The capacity of transgenic tobacco to send a systemic RNA silencing signal depends on the nature of the inducing transgene locus

RNA silencing is a conserved eukaryotic pathway in which double-stranded RNA (dsRNA) triggers destruction of homologous target RNA via production of short-interfering RNA (siRNA). In plants, at least some cases of RNA silencing can spread systemically. The signal responsible for systemic spread is expected to include an RNA component to account for the sequence specificity of the process, and transient silencing assays have shown that the capacity for systemic silencing correlates with the accumulation of a particular class of small RNA. Here, we report the results of grafting experiments to study transmission of silencing from stably transformed tobacco lines in the presence or absence of helper component-proteinase (HC-Pro), a viral suppressor of silencing. The studied lines carry either a tail-to-tail inverted repeat, the T4-IR transgene locus, or one of two different amplicon transgene loci encoding replication-competent viral RNA. We find that the T4-IR locus, like many sense-transgene-silenced loci, can send a systemic silencing signal, and this ability is not detectably altered by HC-Pro. Paradoxically, neither amplicon locus effectively triggers systemic silencing except when suppressed for silencing by HC-Pro. In contrast to results from transient assays, these grafting experiments reveal no consistent correlation between capacity for systemic silencing and accumulation of any particular class of small RNA. In addition, although all transgenic lines used to transmit systemic silencing signals were methylated at specific sites within the transgene locus, silencing in grafted scions occurred without detectable methylation at those sites in the target locus of the scion.     

Localisation and movement of Plum pox virus in apricot stem tissues

Localisation and movement of Plum pox virus (PPV), sharka disease, in stem tissues of susceptible and resistant apricot (Prunus armeniaca L.) cultivars was studied. Two different assays were performed. In the first assay, apricot cultivars were grafted on to a non‐inoculated GF305 peach rootstock and, after two months, the sprouted apricot was inoculated by chip‐grafting. In the second assay, apricot cultivars were grafted on to a previously chip‐inoculated GF305 showing strong PPV symptoms. Localisation of virus was studied in apricot stem by immuno‐tissue printing and sharka symptoms in GF305 and apricot leaves were also observed. Virus was mainly localised in the xylem, and sometimes in the cortex and pith. Results revealed that, while all the cultivars allowed limited virus movement from the inoculation point, only the susceptible cultivars (Screara, Bebeco and Colomer) allowed long distance movement and even showed symptoms in leaves.     

Prevalence of avian pox virus and effect on the fledging success of Laysan Albatross

ABSTRACT.   Avian pox virus ( Poxvirus avium ) is a mosquito-borne disease that occurs worldwide in a variety of bird species, but little is known about its prevalence or effect on seabirds. We monitored prevalence of pox virus and its effect on fledging success of Laysan Albatross ( Phoebastria immutabilis ) on Oahu, Hawaii, from 2003 to 2007. Pox prevalence in albatross chicks averaged 88% in years with high rainfall and 3% in years with low rainfall. Diagnosis of pox virus was clinically confirmed in two birds by Muscovy Duck ( Cairina moschata ) fibrolast cultures. Severity of infection ranged from small wart-like nodules and lesions on the bill, face, eyes, tarsus, and feet, to large tumorous growths that completely covered both eyes and caused deformation of the bill and skull. Most chicks recovered from infection, and the fledging rate in pox epizootic years (82%) did not differ from that in years with low pox prevalence (80%) or the average fledging rate on Midway Atoll (86%). Three chicks with severe infections were resighted as healthy adults on Kauai and Oahu in 2007, confirming postfledging survival of at least some birds. The high recovery rate, fledging success, and postfledging survival indicate that Laysan Albatross have strong immunity to avian pox virus.     

Signaling requirements and role of salicylic acid in HRT- and rrt-mediated resistance to turnip crinkle virus in Arabidopsis

Inoculation of turnip crinkle virus (TCV) on the resistant Arabidopsis ecotype Di-17 elicits a hypersensitive response (HR), which is accompanied by increased expression of pathogenesis-related (PR) genes. Previous genetic analyses revealed that the HR to TCV is conferred by HRT, which encodes a coiled-coil (CC), nucleotide-binding site (NBS) and leucine-rich repeat (LRR) class resistance (R) protein. In contrast to the HR, resistance to TCV requires both HRT and a recessive allele at a second locus designated rrt. Here, we demonstrate that unlike most CC-NBS-LRR R genes, HRT/rrt-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also independent of RAR1 and SGT1. HRT/rrt-mediated resistance is compromised in plants with reduced salicylic acid (SA) content as a consequence of mutations eds5, pad4, or sid2. By contrast, HR is not affected by mutations in eds1, eds5, pad4, sid2, ndr1, rar1, or sgt1b. Resistance to TCV is restored in both SA-deficient Di-17 plants expressing the nahG transgene and mutants containing the eds1, eds5, or sid2 mutations by exogenous application of SA or the SA analog benzo(1,2,3)thiadiazole-7-carbothioic acid (BTH). In contrast, SA/BTH treatment failed to enhance resistance in HRT pad4, Col-0, or hrt homozygous progeny of a cross between Di-17 and Col-0. Thus, HRT and PAD4 are required for SA-induced resistance. Exogenously supplied SA or high endogenous levels of SA, due to the ssi2 mutation, overcame the suppressive effects of RRT and enhanced resistance to TCV, provided the HRT allele was present. High levels of SA upregulate HRT expression via a PAD4-dependent pathway. As Col-0 transgenic lines expressing high levels of HRT were resistant to TCV, but lines expressing moderate to low levels of HRT were not, we conclude that SA enhances resistance in the RRT background by upregulating HRT expression. These data suggest that the HRT-TCV interaction is unable to generate sufficient amounts of SA required for a stable resistance phenotype, and the presence of rrt possibly corrects this deficiency.     

Targeting of genomic and negative‐sense strands of viral RNA contributes to antiviral resistance mediated by artificial miRNAs and promotes the emergence of complex viral populations

Technology based on artificial small RNAs, including artificial microRNAs (amiRNAs), exploits natural RNA silencing mechanisms to achieve silencing of endogenous genes or pathogens. This technology has been successfully employed to generate resistance against different eukaryotic viruses. However, information about viral RNA molecules effectively targeted by these small RNAs is rather conflicting, and factors contributing to the selection of virus mutants escaping the antiviral activity of virus‐specific small RNAs have not been studied in detail. In this work, we transformed Nicotiana benthamiana plants with amiRNA constructs designed against the potyvirus plum pox virus (PPV), a positive‐sense RNA virus, and obtained lines highly resistant to PPV infection and others showing partial resistance. These lines have allowed us to verify that amiRNA directed against genomic RNA is more efficient than amiRNA targeting its complementary strand. However, we also provide evidence that the negative‐sense RNA strand is cleaved by the amiRNA‐guided RNA silencing machinery. Our results show that the selection pressure posed by the amiRNA action on both viral RNA strands causes an evolutionary explosion that results in the emergence of a broad range of virus variants, which can further expand in the presence, and even in the absence, of antiviral challenges.     

Induction of systemic resistance in tobacco against Tobacco mosaic virus by Bacillus spp.

Bacillus pumilus strain EN16 and Bacillus subtilis strain SW1 were tested for their systemic resistance and protection abilities against tobacco mosaic virus disease under greenhouse conditions. The results showed that strain EN16 and SW1 treatment significantly reduced mosaic symptoms and disease severity, resulting in 52 and 71% protection at 14 days of inoculation, respectively. A decreased amount of virus was detected in EN16- or SW1-treated tobacco plants by ELISA. Moreover, 5- and 7-day intervals between inducer treatment and pathogen inoculations were respectively required for strain EN16 and SW1 to induce optimal resistance. Further analysis on phenylalanine ammonia-lyase, peroxidase, polyphenol oxidase and pathogenesis-related (PR) proteins in tobacco showed that the amounts of defense enzymes and PR proteins significantly increased in Bacillus-treated plants challenged with pathogen when compared to control.     

Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana

Eukaryotes employ RNA silencing as an innate defense system against invading viruses. Dicer proteins play the most crucial role in initiating this antiviral pathway as they recognize and process incoming viral nucleic acids into small interfering RNAs. Generally, 2 successive infection stages constitute viral infection in plants. First, the virus multiplies in initially infected cells or organs after viral transmission and then the virus subsequently spreads systemically through the vasculature to distal plant tissues or organs. Thus, antiviral silencing in plants must cope with both local and systemic invasion of viruses. In a recent study using 2 sets of different experiments, we clearly demonstrated the differential requirement for Dicer-like 4 (DCL4) and DCL2 proteins in the inhibition of intracellular and systemic infection by potato virus X in Arabidopsis thaliana. Taken together with the results of other studies, here we further discuss the functional specificity of DCL proteins in the antiviral silencing pathway.