Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling.

Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.

Genotoxicity evaluation of effluents from textile industries of the region Fez-Boulmane, Morocco: a case study.

In order to investigate the biological hazard of effluents from textile industries of Fez-Boulmane region in Morocco, mutagenicity and phytotoxicity tests were performed on different biological systems. Moreover, the efficiency of a Sequencing Batch Reactor (SBR) system, working by activated sludge on a laboratory scale, was estimated by comparing the ecotoxicity results observed before and after wastewater treatment. Evaluation of the genotoxic potential was investigated by means of classic mutagenicity tests on D7 strain of Saccharomyces cerevisiae and by phytotoxicity tests on Allium sativum L., Vicia faba L. and Lactuca sativa L., estimating micronuclei presence, mitotic index and cytogenetic anomalies. The results obtained by testing untreated wastewater demonstrated major genotoxicity effects in S. cerevisiae and various levels of phytotoxicity in the three plant systems, while after SBR treatment no more ecotoxicological consequences were observed. These data confirm the effectiveness of the SBR system in removing toxic substances from textile wastewaters in Fez-Boulmane region.

Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement.

We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.

Cauliflower mosaic virus protein P6 inhibits signaling responses to salicylic acid and regulates innate immunity.

Cauliflower mosaic virus (CaMV) encodes a multifunctional protein P6 that is required for translation of the 35S RNA and also acts as a suppressor of RNA silencing. Here we demonstrate that P6 additionally acts as a pathogenicity effector of an unique and novel type, modifying NPR1 (a key regulator of salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling) and inhibiting SA-dependent defence responses We find that that transgene-mediated expression of P6 in Arabidopsis and transient expression in Nicotiana benthamiana has profound effects on defence signaling, suppressing expression of representative SA-responsive genes and increasing expression of representative JA-responsive genes. Relative to wild-type Arabidopsis P6-expressing transgenics had greatly reduced expression of PR-1 following SA-treatment, infection by CaMV or inoculation with an avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst). Similarly transient expression in Nicotiana benthamiana of P6 (including a mutant form defective in translational transactivation activity) suppressed PR-1a transcript accumulation in response to Agrobacterium infiltration and following SA-treatment. As well as suppressing the expression of representative SA-regulated genes, P6-transgenic Arabidopsis showed greatly enhanced susceptibility to both virulent and avirulent Pst (titres elevated 10 to 30-fold compared to non-transgenic controls) but reduced susceptibility to the necrotrophic fungus Botrytis cinerea. Necrosis following SA-treatment or inoculation with avirulent Pst was reduced and delayed in P6-transgenics. NPR1 an important regulator of SA/JA crosstalk, was more highly expressed in the presence of P6 and introduction of the P6 transgene into a transgenic line expressing an NPR1:GFP fusion resulted in greatly increased fluorescence in nuclei even in the absence of SA. Thus in the presence of P6 an inactive form of NPR1 is mislocalized in the nucleus even in uninduced plants. These results demonstrate that P6 is a new type of pathogenicity effector protein that enhances susceptibility to biotrophic pathogens by suppressing SA- but enhancing JA-signaling responses.

Characterization of LEAFY COTYLEDON1-LIKE gene in Helianthus annuus and its relationship with zygotic and somatic embryogenesis.

The Helianthus annuus LEAFY COTYLEDON1-LIKE (HaL1L) gene encodes a heme-activated protein 3 subunit of the CCAAT box-binding factor. The phylogenetic analysis indicates that HaL1L is closely related to LEAFY COTYLEDON1 (LEC1)-type of Arabidopsis thaliana. In particular, the peptide results homologous to the LEC1-LIKE gene of A. thaliana, with which it shares a high amino acid sequence identity (56%). HaL1L transcripts are accumulated primarily at an early stage of sunflower embryogenesis. High levels of HaL1L messenger RNA (mRNA) have been detected in the developing embryo proper, suspensor, endosperm, integument, and integumentary tapetum cells, while in unfertilized ovules, HaL1L mRNA was present at rather low levels. In an attempt to examine the involvement of HaL1L on somatic embryogenesis, a somaclonal variant of H. annuus x H. tuberosus (EMB-2) that produces ectopic embryo- and shoot-like structures, arranged in clusters along leaf veins, was used. We found that the epiphyllous proliferation of ectopic embryos on EMB-2 leaves was associated to HaL1L mRNA accumulation. The detection of HaL1L transcripts was evident in somatic embryos at the heart- and early cotyledon-stage. On the contrary, no signal related to HaL1L transcript accumulation was observed in EMB-2 leaves characterized by the presence of shoot-like structures. Together, these results support the conclusion that the transcription of the HaL1L gene is maintained both in zygotic and in somatic embryogenesis. In addition, the ectopic accumulation of HaL1L mRNA in parenchymal cells around the vascular bundles of epiphyllous leaves opens the possibility that HaL1L could also be involved in switching somatic cell fate towards embryogenic competence.

Expression of two genes encoding gibberellin 2- and 3-oxidases in developing seeds of Phaseolus coccineus.

We have isolated PcGA3ox1, a cDNA clone from developing runner bean (Phaseolus coccineus) seeds that shows significant amino acid homology with the gibberellin (GA) 3-oxidases. A recombinant fusion protein of PcGA3ox1 converted GA20 and GA9 to GA1 and GA4, respectively. In situ hybridization results showed that transcripts of this gene accumulate specifically within the suspensor of globular-stage embryos. PcGA3ox1 mRNA begins to accumulate in the epidermal cells of the embryo proper and is also detectable in the endosperm during the transition from globular- to heart-stage embryos. PcGA3ox1 transcripts were localized exclusively in the cotyledons from the early cotyledonary stage up to the cotyledonary stage. Transcripts of the previously cloned GA 2-oxidase (PcGA2ox1) from developing seeds of runner bean were found primarily within the suspensor neck region from the late globular stage up to the heart stage. PcGA2ox1 mRNA was detectable in the whole suspensor from the early cotyledonary stage, and was found in the inner layer of integuments at the cotyledonary stage. Soluble enzyme preparations made from suspensors and embryos at two stages of embryogenesis (the heart and cotyledonary stages) were incubated with [14C]GA20 and [14C]GA1. Only young suspensor preparations converted GA20 to GA1 and GA5. Both suspensor preparations converted GA1 to GA8. Both embryo preparations converted GA20 to GA1, but were unable to convert GA1 to GA8.

Mutations that delay flowering in Arabidopsis de-couple symptom response from cauliflower mosaic virus accumulation during infection.

summary The development of disease symptoms in plants infected with a compatible virus involves complex signalling interactions between host and viral gene products. Photoperiod is an important influence on the transition from vegetative growth to flowering. Symptoms in wild-type Arabidopsis plants grown under long days were much less severe than in plants grown under short days, although under long days, the levels of replicating virus were 1.5-1.8 times greater than in plants grown in short days. We tested the effects on response to CaMV infection of mutations at two of the loci that control the transition from vegetative growth to flowering, FCA and GI. In long days, CaMV-infected fca-1 mutants and strong gi alleles developed much more severe symptoms than wild-type. Despite the increased symptom severity, levels and distribution of replicating CaMV in fca-1 and gi mutants were similar to those in wild-type. In short days, both mutants and wild-type grew vegetatively. Virus accumulation and symptom developments in fca-1 were similar to the wild-type, but in strong gi alleles, symptom progression in apical leaves was very delayed, although virus accumulation was similar to the wild-type controls. The developmental state of the plants influences the symptom response; however, it does not appear to do so by directly effecting overall virus titre or distribution. The altered symptom response of gi mutants in short days suggests an additional role for GI. These mutants provide compelling evidence for the existence of specific pathways for disease signalling.

Arabidopsis mutants that suppress the phenotype induced by transgene-mediated expression of cauliflower mosaic virus (CaMV) gene VI are less susceptible to CaMV-infection and show reduced ethylene sensitivity.

Protein P6 is the main symptom determinant of cauliflower mosaic virus (CaMV), and transgene-mediated expression in Arabidopsis induces a symptom-like phenotype in the absence of infection. Seeds of a P6-transgenic line, A7, were mutagenized by gamma-irradiation and M2 seedlings were screened for mutants that suppressed the phenotype of chlorosis and stunting. We identified four mutants that were larger and less chlorotic than the A7 parent but which contained an intact and transcriptionally active transgene. The two mutants with the strongest suppression phenotype, were recessive and allelic. The transgene was eliminated by back-crossing with wild-type Arabidopsis. In progeny lines that were homozygous for the putative suppressor mutation the proportion of plants becoming infected following inoculation with CaMV was 40% that of wild-type, although in plants that did become infected, levels of virus DNA in mutants and wild-type did not differ significantly. Symptoms in the mutants were milder and delayed although this was somewhat dependent on the virus isolate. This phenotype was inherited stably. Both mutant alleles showed a partially ethylene-insensitive phenotype in an ethylene triple response assay. P6-transgenic plants were also almost completely insensitive to ethylene in the triple response assay. We suggest that the chlorosis and stunting in P6-transgenic and CaMV-infected plants are dependent on interactions between P6 and components involved in ethylene signalling, and that the suppressor gene product may function to augment these interactions.