Host cell entry of powdery mildew is correlated with endosomal transport of antagonistically acting VvPEN1 and VvMLO to the papilla.

Challenge by a nonadapted powdery mildew fungal pathogen leads to the formation of a local cell-wall apposition (papilla) beneath the point of attempted penetration. Several plasma membrane (PM) proteins with opposing roles in powdery mildew infection, including Arabidopsis thaliana PENETRATION1 (PEN1) and barley (Hordeum vulgare) MILDEW RESISTANCE LOCUS O (MLO), are localized to the site of powdery mildew attack. PEN1 contributes to penetration resistance to nonadapted powdery mildews, whereas MLO is a susceptibility factor required by adapted powdery mildew pathogens for host cell entry. Our previous studies have demonstrated that the vesicle and endosomal trafficking inhibitors, brefeldin A and wortmannin, have opposite effects on the penetration rates of adapted and nonadapted powdery mildews on grapevine. These findings prompted us to study the pathogen-induced intracellular trafficking of grapevine variants of MLO and PEN1. We first identified grapevine (Vitis vinifera) VvPEN1 and VvMLO orthologs that rescue Arabidopsis Atpen1 and Atmlo2 mlo6 mlo12 null mutants, respectively. By using endomembrane trafficking inhibitors in combination with fluorescence microscopy, we demonstrate that VvMLO3/VvMLO4 and VvPEN1 are co-trafficked together from the PM to the site of powdery mildew challenge. This focal accumulation of VvMLO3/VvMLO4 and VvPEN1 to the site of attack seems to be required for their opposing functions during powdery mildew attack, because their subcellular localization is correlated with the outcome of attempted powdery mildew penetration.

Characterization and transient replication of tomato leaf curl virus defective DNAs.

Distinct subgenomic DNA species known as defective (df) DNA molecules were found in plants infected with tomato leaf curl virus (TLCV). Four df DNAs derived from TLCV Type and Darwin 1 strains were found to contain large deletions that disrupt all of the viral genes required for viral replication, encapsidation and spread. However, the viral origin of replication (ori), including the replication-associated protein (Rep) binding domains, was present in all four df DNAs. Co-agroinfection of leaf strips with tandem repeat constructs of the viral and df DNAs resulted in their replication in the presence of the respective TLCV strain. However, the df DNAs failed to move in whole plants when co-inoculated with TLCV. The df DNAs were shown to be associated with TLCV coat protein, which may indicate encapsidation. Mutational analysis showed the minimum sequence requirements for df DNA replication by TLCV to be the intergenic region containing the Rep-binding domains.

Measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralising antibodies in mice following mucosal vaccination.

Plant-made oral vaccines have the potential to overcome many of the limitations of traditional vaccines. Here we report on progress towards a lettuce-made measles vaccine. Lettuce is a palatable species which exhibits rapid growth in contained hydroponic systems and produces negligible quantities of toxins. Measles virus hemagglutinin (MV-H) protein was successfully expressed in transgenic lettuce and found to be immunogenic in mice. Lettuce extracts containing MV-H protein induced MV neutralising antibodies following intraperitoneal injection and intranasal inoculation of mice. Using a sequential prime-boost strategy in which mice were vaccinated with MV-H DNA followed by an orally delivered freeze-dried MV-H lettuce formulation a 10-fold increased in MV-specific IgG titers was observed relative to mice vaccinated with control lettuce formulations (p=0.05). MV-H protein was stable in freeze-dried lettuce for up to 13 months at room temperature, and survived at least a week at temperatures as high as 50 degrees C. This research represents a significant step towards the development of measles vaccine formulation that is effective, temperature-stable, easy to administer in a resource-poor setting and amenable to large scale manufacture.

Identification of resistance gene analogs linked to a powdery mildew resistance locus in grapevine.

Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogs (RGAs) from grapevine. Twenty eight unique grapevine RGA sequences were identified and subdivided into 22 groups on the basis of nucleic acid sequence-identity of approximately 70% or greater. Representatives from each group were used in a bulked segregant analysis strategy to screen for restriction fragment length polymorphisms linked to the powdery mildew resistance locus, Run1, introgressed into Vitis vinifera L. from the wild grape species Muscadinia rotundifolia. Three RGA markers were found to be tightly linked to the Run1 locus. Of these markers, two (GLP1-12 and MHD145) cosegregated with the resistance phenotype in 167 progeny tested, whereas the third marker (MHD98) was mapped to a position 2.4 cM from the Run1 locus. The results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species.

A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF, is not only expressed in flowers but also throughout berry development.

An AGAMOUS/SHATTERPROOF homologue (Vvmads1) was isolated from grapevine by differential display between berry and leaf mRNA. The predicted protein sequence of the full-length clone shows a high degree of homology to PLENA (77% identity) and to SHP1 and SHP2 (75% and 74% identity respectively), and is grouped with AGAMOUS/PLENA homologues when the conserved MADS and K domains are compared. Vvmads1 is expressed only in the later stages of flower development and throughout berry development, although expression is reduced after ripening commenced. When Vvmads1 was over-expressed in tobacco, the resulting plants display altered morphologies in the outer two floral whorls. In the most extreme cases, the inner whorls were surrounded by a carpelloid structure created by the modified sepals. Within these sepals were petals which had been split into sections and which were attached at the base of the flower by structures with the appearance of filaments. The results of this study suggest that Vvmads1 has a regulatory role in flower development before fertilisation and a role in fruit development after fertilisation.

The production of an inducible antisense alternative oxidase (Aox1a) plant.

Plant mitochondria contain an alternative oxidase (AOX) acting as a terminal electron acceptor of the alternative pathway in the electron transport chain. Here we describe the production of inducible antisense Aox1a plants of Arabidopsis thaliana (L.) Heynh. and the procedures used to determine the resulting alternative pathway activity. The Arabidopsis Aox1a cDNA sequence was cloned behind a copper-inducible promoter system in the antisense orientation. Arabidopsis thaliana (Columbia) plants were transformed by in-planta vacuum infiltration with Agrobacterium containing the antisense construct. Whole-leaf ethanol production was used as a measure to investigate alternative pathway activity in the presence of antimycin A. After 24 h, leaves from the copper-induced, antisense line F1.1 produced up to 8.8 times more ethanol (via aerobic fermentation) than the non-induced and wild-type leaves, indicating effective cytochrome pathway inhibition by antimycin A and a decreased alternative pathway activity in induced F1.1 leaves. Transgene expression studies also revealed no expression in non-induced leaves and up until 24 h post-induction. Copper-induced transgenic leaves were less susceptible to alternative pathway inhibition than non-induced transgenic leaves, as seen via tissue-slice respiratory studies, and mitochondrial respiration, using F1.1 cell cultures, also supported this. These results demonstrate the successful production of a transgenic line of Arabidopsis in which the alternative pathway activity can be genetically manipulated with an inducible antisense system.

Glycosyl-phosphatidylinositol-anchor addition signals are processed in Nicotiana tabacum.

Recent studies have demonstrated the existence of glycosyl-phosphatidylinositol (GPI)-anchored proteins in higher plants. In this study we tested whether GPI-addition signals from diverse evolutionary sources would function to link a GPI-anchor to a reporter protein in plant cells. Tobacco protoplasts were transiently transfected with a truncated form of the Clostridium thermocellum endoglucanase E reporter gene (celE') fused with a tobacco secretion signal (PR-1a) at the N-terminus and either a yeast (GAS1), mammalian (Thy-1) or putative plant (LeAGP-1) GPI-anchor addition signal at the C-terminus. The yeast and plant C-terminal signals were found to be capable of directing the addition of a GPI-anchor to the endoglucanase protein (EGE') as shown by the sensitivity of the lipid component of GPI to phosphatidylinositol-specific phospholipase C (PI-PLC) digestion. In contrast, the mammalian signal was poorly processed for anchor addition. When EGE' was fused to a truncated form of the LeAGP-1 signal (missing three amino acids predicted to be critical to signal cleavage and anchor addition), a GPI-anchor was not linked to the EGE' protein indicating the necessity for the missing amino acids. Our results show the conservation of the properties of GPI-signals in plant cells and that there may be some similar preferences in GPI-addition signal sequences for yeast and plant cells.

Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus.

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.

A class IV chitinase is highly expressed in grape berries during ripening.

Chitinase activity increased markedly at the onset of ripening in grape (Vitis vinifera L.) berries and continued to increase throughout the sugar accumulation phase of berry development. In contrast, beta-1,3-glucanase activity was not detected in grape berries at any stage of development. Two closely related chitinase cDNAs (VvChi4A and VvChi4B) were cloned from grapes. Sequence and Southern analysis indicate that these two clones may represent alleles of the same gene. The predicted proteins are acidic and have a signal peptide followed by a cysteine-rich, chitin-binding domain and a catalytic region. An analysis of their sequences indicates that they are class IV chitinase. The deduced protein sequence of VvChi4A has a high level of identity with the 32- and 28-kD chitinases present as haze proteins in wine. Expression of VvChi4 was high in berries and low in flowers but was not detected in leaves, roots, or seeds. No expression was detected in berries 2 to 8 weeks postflowering, but expression was high 12 to 16 weeks postflowering, which coincided with sugar accumulation and an increase in chitinase activity. Constitutive expression of VvChi4 appears to be fruit-specific and induced at high levels in grapes during ripening.

A novel subviral agent associated with a geminivirus: the first report of a DNA satellite.

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem-loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication.

Detection of glycosyl-phosphatidylinositol-anchored proteins on the surface of Nicotiana tabacum protoplasts.

Glycosyl-phosphatidylinositol (GPI)-anchored plasma membrane proteins have been found to be widespread in eukaryotes and protozoa but have not been reported in higher terrestrial plants. A sensitive biotin-based assay has been used to detect the presence of GPI-anchored proteins on the outer surface of cultured Nicotiana tabacum cells. Six proteins with molecular weights of 92, 84, 60.5, 54.5, 39.5 and 37 kDa were found to move from a Triton X-114 detergent-rich phase to an aqueous phase following incubation with phosphatidylinositol-specific phospholipase C (PtdIns-PLC). The behaviour of these proteins is consistent with the presence of a GPI-anchor. Seven GPI-anchored proteins were also detected on the surface of tobacco leaf protoplasts with molecular weights of 67.5, 62, 39, 33.5, 27, 23 and 15.6 kDa. These data demonstrate the presence of multiple GPI-anchored proteins on the plasma membrane of higher plant cells.

Nucleotide sequence of grapevine (Vitis vinifera) cDNA similar to SNAP proteins.

A cDNA clone (VS1) homologous to SNAP proteins was isolated from a grapevine cDNA library. The cDNA insert was 1167 bp long and contained a single open reading frame coding for 289 amino acids. The amino acid sequence of VS1 shows similarity (35%-45%) to SNAP proteins from various sources.

Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses?

Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons.

Isolation of a full-length cDNA encoding polyphenol oxidase from sugarcane, a C4 grass.

Polyphenol oxidase (PPO) activity in sugarcane (a C4 grass) was highest in the growing point and declined down the stalk. Sugarcane PPO with an apparent molecular mass of 45 kDa was purified to homogeneity from immature stem tissue. Western analysis of sugarcane extracts with a polyclonal antibody raised to this protein suggested it resulted from cleavage of a 60 kDa protein during purification. The antibody was used to screen a sugarcane stem cDNA library. A full-length PPO clone (sugppo 1) was characterised and shown to encode a 67 kDa precursor protein comprising a plastid transit sequence of 8 kDa and a mature PPO protein of 59 kDa. High levels of expression of sugppo 1 were detected in the growing point of the stalk and in the immature tissue immediately below it, but no message was detected in RNA from mature stem or leaf. Comparison with other PPO sequences indicated that sugppo 1 was significantly different to PPO genes in C3 dicotyledonous plants.

Polyphenol oxidase in potato. A multigene family that exhibits differential expression patterns.

Polyphenol oxidase (PPO) activity in potato (Solanum tuberosum) plants was high in stolons, tubers, roots, and flowers but low in leaves and stems. PPO activity per tuber continued to increase throughout tuber development but was highest on a fresh weight basis in developing tubers. PPO activity was greatest at the tuber exterior, including the skin and cortex tissue 1 to 2 mm beneath the skin. Flowers had high PPO activity throughout development, particularly in the anthers and ovary. Five distinct cDNA clones encoding PPO were isolated from developing tuber RNA. POT32 was the major form expressed in tubers and was found in all parts of the tuber and at all stages of tuber development. It was also expressed in roots but not in photosynthetic tissues. POT33 was expressed in tubers but mainly in the tissue near the skin. POT72 was detected in roots and at low levels in developing tubers. NOR333 was identical with the P2 PPO clone previously isolated from potato leaves (M.D. Hunt, N.T. Eannetta, Y. Haifeng, S.M. Newman, J.C. Steffens [1993] Plant Mol Biol 21: 59-68) and was detected in young leaves and in tissue near the tuber skin but was highly expressed in flowers. The results indicate that PPO is present as a small multigene family in potato and that each gene has a specific temporal and spatial pattern of expression.

The termini of a new citrus viroid contain duplications of the central conserved regions from two viroid groups.

A citrus viroid associated with dwarfing, CVdIIIA, has been sequenced and its 294 nucleotide residues can be arranged to form the typical rod-like secondary structure of other viroids with 71% of nucleotides base-paired. CVdIIIA has greatest sequence similarity with apple scar skin viroid (ASSVd; 69%) and has the central sequence which is conserved in the ASSVd group. CVdIIIA is the smallest member of the ASSVd group but contains the terminal conserved region shared by all viroids over 300 nucleotides. The two ends of CVdIIIA are highly unusual in that each end appears to be derived from the conserved central core region of a different viroid group.

ORF C4 of tomato leaf curl geminivirus is a determinant of symptom severity.

A tomato leaf curl virus (TLCV) mutant has been constructed in vitro that contains T-to-C replacements at nucleotides 2457 and 2463 within the C4 open reading frame (ORF). The mutations destroy the two possible initiator AUG codons for the C4 ORF without disrupting the coding capacity of the C1 ORF which entirely overlaps the C4 ORF. Agroinoculation of the C4 mutant TLCV into three alternative experimental hosts for the virus (Datura stramonium, Lycopersicon esculentum, and Nicotiana tabacum) gives rise to infections which show dramatically reduced symptoms when compared to a wild-type infection, while retaining wild-type levels of all viral DNA species. In most cases the mutations were stably inherited by progeny virus. However, a single tomato plant inoculated with the mutant developed phenotypically wild-type symptoms and was subsequently shown to contain progeny virus in which the mutation at position 2457 had reverted to wild-type sequence, indicating that this AUG may be the site of initiation of translation of the C4 product in the wild-type virus. The results suggest that the C4 ORF encodes a polypeptide which is not required by TLCV to replicate or to spread through the host plant, but is involved in symptom development.

Molecular cloning and characterisation of grape berry polyphenol oxidase.

Polyphenol oxidase (PPO) was purified to homogeneity from Sultana grapes yielding a single protein with an apparent molecular mass of 40 kDa as determined by SDS-PAGE. A degenerate oligonucleotide primer based on the N-terminal amino acid sequence of this purified 40 kDa grape PPO protein was used to amplify a 1650 bp cDNA clone (GPO1M) by 3' rapid amplification of cDNA ends (3'-RACE). GPO1M hybridized to a single 2.2 kb transcript from grape berry mRNA indicating the presence of further upstream sequence which was cloned using 5'-RACE PCR. The complete 1990 bp cDNA (GPO1) encodes a 67 kDa protein consisting of a 10.6 kDa chloroplast transit peptide, a 40.5 kDa catalytic unit containing two copper-binding regions and a 16.2 kDa C-terminal extension. Southern analysis suggested the presence of only one PPO gene in grapevine. High levels of gene expression were found in young developing berries, leaves and roots, but there was little expression in mature tissues. Biogenesis of PPO in grapevine tissues, appears to involve synthesis of a 67 kDa precursor protein which is imported into the chloroplast and processed to remove a 10.6 kDa chloroplast transit peptide from the N-terminus and a 16.2 kDa peptide of unknown function from the C-terminus.

Mutagenesis of the virion-sense open reading frames of tomato leaf curl geminivirus.

A series of frame shift, deletion, and inversion mutants in the virion-sense open reading frames (ORFs) of the monopartite geminivirus tomato leaf curl virus have been constructed and their ability to replicate, produce single-stranded DNA, spread, and cause symptoms in tomato plants has been investigated. Disruptions in the V1 ORF lead to symptomless, systemic infections with a reduced titer of all viral DNA forms while interruptions in the V2 (coat protein) ORF disrupted spread of the virus. Mutagenesis of the virion-sense ORFs did not affect the replication of viral double-stranded DNA, although both V1 and V2 products appear to play a role in the accumulation of viral single-stranded DNA.

Mapping of the polycistronic RNAs of tomato leaf curl geminivirus.

Four major transcripts from infected tomato were identified and mapped onto the monopartite genome of the geminivirus tomato leaf curl virus (TLCV). The C1, C2, and C3 ORFs are spanned by one transcript and a second internal RNA covered C2 and C3. Both these RNAs have their counterparts in the DNA A components of the bipartite subgroup of geminiviruses. The 5' ends of the virion-sense RNAs map either side of the first in-frame AUG of the V1 ORF. The 3' ends of the virion-sense RNAs are coterminal and overlap with the 3' ends of the complementary-sense RNAs. All of the RNAs have transcription regulatory sequences close to their mapped termini and the presence of overlapping transcripts suggests that temporal regulation of their synthesis may occur. The translation of these polycistronic RNAs is discussed in the light of the RNA mapping data.