Nadia S. Al-Kaff

Detailed Dissection of the Chromosomal Region Containing the Ph1 Locus in Wheat Triticum aestivum: With Deletion Mutants and Expression Profiling

Background and Aims Understanding Ph1, a dominant homoeologous chromosome pairing suppressor locus on the long arm of chromosome 5B in wheat Triticum aestivum L., is the core of the investigation in this article. The Ph1 locus restricts chromosome pairing and recombination at meiosis to true homologues. The importance of wheat as a crop and the need to exploit its wild relatives as donors for economically important traits in wheat breeding programmes is the main drive to uncover the mechanism of the Ph1 locus and regulate its activity. Methods Following the molecular genetic characterization of the Ph1 locus, five additional deletion mutants covering the region have been identified. In addition, more bacterial artificial chromosomes (BACs) were sequenced and analysed to elucidate the complexity of this locus. A semi-quantitative RT–PCR was used to compare the expression profiles of different genes in the 5B region containing the Ph1 locus with their homoeologues on 5A and 5D. PCR products were cloned and sequenced to identify the gene from which they were derived. Key Results Deletion mutants and expression profiling of genes in the region containing the Ph1 locus on 5B has further restricted Ph1 to a cluster of cdk-like genes. Bioinformatic analysis of the cdk-like genes revealed their close homology to the checkpoint kinase Cdk2 from humans. Cdk2 is involved in the initiation of replication and is required in early meiosis. Expression profiling has revealed that the cdk-like gene cluster is unique within the region analysed on 5B in that these genes are transcribed. Deletion of the cdk-like locus on 5B results in activation of transcription of functional cdk-like copies on 5A and 5D. Thus the cdk locus on 5B is dominant to those on 5A and 5D in determining the overall activity, which will be dependent on a complex interplay between transcription from non-functional and functional cdk-like genes. Conclusions The Ph1 locus has been defined to a cdk-like gene cluster related to Cdk2 in humans, a master checkpoint gene involved in the initiation of replication and required for early meiosis.

Plants rendered herbicide-susceptible by cauliflower mosaic virus-elicited suppression of a 35S promoter-regulated transgene

Crop plants genetically modified for herbicide tolerance were some of the first to be released into the environment. Frequently, the cauliflower mosaic virus (CaMV) 35S promoter is used to drive expression of the herbicide tolerance transgene. We analyzed the response to CaMV infection of a transgenic oilseed rape line containing the bialaphos tolerance gene (BAR) from Streptomyces hygroscopicus, regulated by the 35S promoter. Oilseed rape is susceptible to CaMV, but plants recover from infection. CaMV infection altered the expression of the herbicide tolerance gene such that plants became susceptible to the herbicide. The effect on transgene expression differed in infections with viral pathogenic variants typical of those found in natural situations worldwide. Susceptibility to the herbicide was most likely a result of transcriptional gene silencing of the transgene. Our results show that transgene phenotypes can be modified by pathogen invasion.

Plant DNA viruses and gene silencing.

Gene silencing is a multifaceted phenomenon leading to propagative down-regulation of gene expression. Gene silencing, first observed in plants containing transgenes, can operate both at the transcriptional and post-transcriptional levels. Silencing effects can be triggered by nuclear transgenes and by cytoplasmic RNA viruses, and it can be propagated between these elements and endogenous plant genes that share sequence homology. Although some aspects of gene silencing are becoming better understood, little is yet known about the relationship between nuclear and cytoplasmic events. Plant DNA viruses – both the ssDNA geminiviruses and the reverse-transcribing pararetroviruses – have properties with the potential to initiate gene silencing in the nucleus and in the cytoplasm. Characteristics include production of multiple copies of viral DNA genomes in the nucleus, illegitimate integration of viral DNA into host chromosomes mimicking transgene transformation, and generation of abundant viral RNAs in the cytoplasm. Evidence is emerging that geminiviruses and plant pararetroviruses can interact with the gene silencing system either from introduced DNA constructs or during viral pathogenesis. Some observations suggest there are complex relationships between DNA viral activity, transcriptional and post-transcriptional gene silencing mechanisms. DNA viruses also have properties consistent with an ability to counteract the plant silencing response. In this article, features of plant DNA viruses are discussed in relation to gene silencing phenomena, and the prospects for understanding the interaction between nuclear and cytoplasmic silencing processes.

Structural and functional relationship between the Ph1 locus protein 5B2 in wheat and CDK2 in mammals

The Ph1 locus in hexaploid wheat is responsible for restricting chromosome pairing at meiosis to true homologues by suppressing homoeologous pairing. Based on detailed modelling studies and predicted ability to form complexes with cyclin-A and cyclin-dependent kinase inhibitor such as p27, Triticum aestivum-5B2 (Ta5B2) is suggested to be a wheat analogue of human CDK2 enzyme. A blast analysis of the protein data bank using the amino acid sequence of the protein expressed by the 5B2 copy of the cdk-like cluster of genes at the Ph1 locus (Ta5B2) identified humans CDK2 as a top hit. In this analysis, the canonical cyclin binding motif PSTAIRE of CDK2 is replaced by a novel DARTLRE motif and Thr160 residue, phosphorylation of which is required for positive regulation of CDK2, is replaced by a tyrosine (Tyr174) in Ta5B2. Despite these differences, detailed analyses show that all residues known to be important for cyclin binding are either fully conserved or whenever there is alteration in Ta5B2, a corresponding but comparable alteration is also observed in plant cyclins notably cyclin-A of Arabidopsis thaliana. Moreover, the Thr160/Tyr174 substitution is also accommodated by suitable alterations in the 3D space around Tyr174 and the 3D model of Ta5B2 predicts Tyr174 to play the same role as Thr160 plays in CDK2.

The molecular features of chromosome pairing at meiosis: the polyploid challenge using wheat as a reference

During meiosis, chromosome numbers are halved, leading to haploid gametes, a process that is crucial for the maintenance of a stable genome through successive generations. The process for the accurate segregation of the homologues starts in pre-meiosis as each homologue is replicated and the respective products are held together as two sister chromatids via specific cohesion proteins. At the start of meiosis, each chromosome must recognise its homologue from amongst all the chromosomes present in the nucleus and then associate or pair with that homologue. This process of homologue recognition in meiosis is more complicated in polyploids because of the greater number of related chromosomes. Despite the presence of these related chromosomes, for polyploids such as wheat to produce viable gametes, they must behave as diploids during meiosis with only true homologues pairing. In this review, the relationship between the Ph1 cyclin-dependent kinase (CDK)-like genes in wheat and the CDK2 genes in mammals and their involvement in controlling this process at meiosis is examined.

Variation in Biological Properties of Cauliflower Mosaic Virus Clones

Infectious clones were prepared from virion DNA of three cauliflower mosaic virus (CaMV) isolates, 11/3, Xinjiang (XJ), and Aust, to investigate pathogenic variation in virus populations. Of 10 infectious clones obtained for isolate 11/3, four pathotypes were identified, each producing symptoms in turnip that differed from those of the 11/3 wild-type. Virus from two clonal groups of 11/3 was transmissible by aphids whereas that from two others was not. Of the five infectious clones obtained from isolate XJ, two groups were identified, one of which differed symptomatically from the wild-type. Only one infectious clone was obtained from isolate Aust and this had properties similar to the wild-type. Restriction enzyme polymorphisms were found in some clonal groups and these correlated with symptoms. Other groups with different pathogenic properties could not be distinguished apart by restriction site polymorphisms. Further variation was observed in the nucleotide sequences of gene II (coding for aphid transmission factor) from these viruses as compared with other CaMV isolates. In the aphid non-transmissible clones of isolate 11/3, one had a Gly to Arg mutation in gene II similar to that of other non-deleted non-transmissible CaMV isolates. The second had a 322 bp deletion at the site of a small direct repeat similar to that of isolate CM4-184 although occurring in a different position. The gene II deletion of isolate 11/3 produced a frame-shift that separated genes II and III by 60 bp. Most CaMV clones studied remained biologically stable producing similar symptoms during subsequent passages. However, one clone (11/3-7) produced two new biotypes during its first passage suggesting that it was relatively unstable. Our results show that wild-type populations of CaMV contain a range of infectious genome variants with contrasting biological properties and differing stability. We suggest that a variety of significant viral phenotypic changes can occur during each infection cycle resulting from relatively small genome changes.

Pararetrovirus–crucifer interactions: attack and defence or modus vivendi?

Abstract The compatible infection of plants by viruses usually leads to the development of systemic symptoms. Symptom expression of this kind is generally understood to be a host response that indicates an inability of the host to defend itself from attack. We have been studying compatible interactions between the plant pararetrovirus cauliflower mosaic virus (CaMV) and its crucifer hosts in order to understand the relationship between viral activity, symptom expression and plant defence. A CaMV protein (P6) appears to play a major role in eliciting symptom expression. This host response leads to a regulation of the viral multiplication cycle that is associated with leaf mosaics. The host regulation of CaMV appears to operate at the transcriptional level through an effect on the 35S promoter, or at the post-transcriptional level by a process that is akin to gene silencing, and can lead to host recovery depending upon the genetic background of the host. The plant apex is a focus for antiviral defence mechanisms, presumably because viral infection of the apical meristem would rapidly compromise the ability of the plant to generate new leaves and flowers for reproduction. The balance of interactions between CaMV and crucifers can provide a sustainable source of host plants to ensure viral propagation and viral exposure allows the host to adapt and develop its repertoire of defence mechanisms.

Caulimovirus Isolation and DNA Extraction

Members of the cauhmovn-us group (1) each have a circular double-stranded DNA genome of approx 8 kbp that is encapsidated m a spherical, naked nucleocapsid of approx 50 nm diameter (Fig. 1). Caulimovnuses charactertsttcally produce subcellular mclusron bodies m infected tissues that contam most of the virions found in cells, embedded m an apparently random manner. The host ranges of mdtvldual caultmovnuses tend to be restricted to one or a few plant families, and group members are transmitted between plants by aphrd vectors Based on possession of all, or most, of these charactertstrcs, 12 defimte, and 3 possible, members of the group have been identified (2) The best-characterized and type member of the cauhmovu-uses IS cauliflower mosaic virus (CaMV), from which the group name derives. The complete nucleotrde sequence of at least eight different CaMV isolates (3-ZO), and that of four other caultmovn-uses (21-14) has been determmed. The orgamzatton of vu-al genes (Fig. 1) IS mostly conserved m sequenced caulimovn-uses, but one member, cassava vein mosaic virus (CVMV), IS somewhat different from the others (14). Repltcatton of cauhmovnuses mvolves alternation of genomes as DNA and RNA forms, progeny vlrron DNA being generated by reverse transcrtptton of a terminally redundant, genome-length RNA uttltzmg a vn-usencoded polymerase This feature IS shared by another group of plant DNA vtruses, the badnavnuses, and by animal hepatms B vn-uses. Such vu-uses have been termed pararetrovnuses to dtstmgutsh them from animal retrovuuses, which package an RNA form of the genome derived by transcrtptton of an integrated provn-us. Sequence homologtes m putative coding regions of dtfferent cauhmovu-uses are relatively low One short sequence 1s conserved among

Multiple pathogenic factors influence aphid transmission of cauliflower mosaic virus from infected plants

Aphid transmission of cauliflower mosaic virus (CaMV) is mediated by a polypeptide (P18) encoded by the viral gene II. We have investigated the factors which influence acquisition by aphids of CaMV variants from infected plants. Aphid non-transmissible (AT-) CaMV isolates with a full-length gene II sequence share two amino acid changes, gly to arg at position 94 and ile to val at 105, relative to wild type transmissible (AT+) isolates. We have mutated the gly to arg at position 94 in the AT+ isolate Cabb B-JI which then exhibited the AT- phenotype as predicted. However, replacement of a DNA fragment in Cabb B-JI with one containing the gly to arg change from the AT- isolate Campbell to produce hybrid pBJIC1 resulted in a change in symptom phenotype as well as in aphid transmissibility. pBJIC1 also showed characteristics of partial transmissibility related to the stage of infection when it was tested. The level of P18 was measured in plants and showed that recombinants based upon the Campbell (AT-) genome accumulated P18 later than those based upon the Cabb B-JI genome (AT+). However, the Campbell P18 or recombinant proteins like it, were still not able to mediate transmission even when the P18 level in plants was relatively high and by employing large numbers of aphids. We conclude that aphid transmissibility of CaMV is influenced by multiple factors including P18 levels, inherent functionality of the protein, pathogenic characters of the infecting strain, and the number of aphids used to test transmissibility.