Colleen M. Higgins

Calcium-Dependent Protein Kinases in Plants: Evolution, Expression and Function

Calcium-dependent protein kinases (CPKs) are plant proteins that directly bind calcium ions before phosphorylating substrates involved in metabolism, osmosis, hormone response and stress signaling pathways. CPKs are a large multigene family of proteins that are present in all plants studied to date, as well as in protists, oomycetes and green algae, but are not found in animals and fungi. Despite the increasing evidence of the importance of CPKs in developmental and stress responses from various plants, a comprehensive genome-wide analysis of CPKs from algae to higher plants has not been undertaken. This paper describes the evolution of CPKs from green algae to plants using a broadly sampled phylogenetic analysis and demonstrates the functional diversification of CPKs based on expression and functional studies in different plant species. Our findings reveal that CPK sequence diversification into four major groups occurred in parallel with the terrestrial transition of plants. Despite significant expansion of the CPK gene family during evolution from green algae to higher plants, there is a high level of sequence conservation among CPKs in all plant species. This sequence conservation results in very little correlation between CPK evolutionary groupings and functional diversity, making the search for CPK functional orthologs a challenge.

Peanut Stripe Potyvirus Resistance in Peanut (Arachis Hypogaea L.) Plants Carrying Viral Coat Protein Gene Sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardment with the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.

Efficient organogenesis of an Australian passionfruit hybrid (Passiflora edulis x Passiflora edulis var. flavicarpa) suitable for gene delivery

An efficient regeneration protocol based on organogenesis from cotyledon explants and suitable for gene delivery has been developed for an Australian passionfruit hybrid. Multiple shoots were regenerated from 30-day-old cotyledon explants on Murashige and Skoog (MS) medium containing 6-benzylvaminopurine (BAP) and coconut water. Media pulsing experiments were conducted to investigate the effect on organogenesis of exposure time of the explants to MS containing 10 mu M BAP and 10% (v/v) coconut water, i.e. passionfruit regeneration medium (PRM). Continuous exposure of these explants to PRM maximised the number of shoots produced to 12.1 per explant. However, periods on hormone-free medium improved the appearance of the shoots and increased the number of explants with shoots from 75 to 84.6%. Further, shoots exposed for 7 days to half-strength MS supplemented with 10 mu M NAA (1-napthalene acetic acid) produced twice as many plantlets than those on half-strength MS alone. Transient GUS histochemical assays indicated delivery of the uidA gene via Agrobacterium tumefaciens.

Evidence for a third taxonomic subgroup of peanut stunt virus from China

On the basis of host reactions and serology, six Chinese peanut stunt virus (PSV) strains were found to be distinct from PSV-E and PSV-W, two type strains representing distinct serological subgroups. Chinese PSV strains were characterized by infecting Chenopodium amaranticolor and C. quinoa systemically. All Chinese strains were serologically closely related to each other, but distinct from PSV-E and more distant from PSV-W. Using two PSV-specific primers designed from conserved regions of the PSV RNA3 nucleotide sequence, cDNA transcribed from RNA3 of two Chinese PSV strains, Mi and S, was amplified by PCR and cloned. The sequenced cDNA of the two PSV strains included 654 nt of the coat protein (CP) gene. The identity of the CP gene nucleotide sequence between PSV-Mi and PSV-S was 99.0%, with 99.5% amino acid identity. Identity of the CP gene nucleotide sequence was 75.6 to 77.8% between PSV-Mi and -S (the two Chinese strains) and PSV-ER and -J in PSV subgroup I; and 74.1 to 74.4% between PSV-Mi and -S, and PSV-W in subgroup II. Based on these results, we propose placing PSV Chinese strains into a new PSV subgroup III.

Sequences of the coat protein gene of five peanut stripe virus (PStV) strains from Thailand and their evolutionary relationship with other bean common mosaic virus sequences

SummaryThe coat protein gene and part of the 3′ non-coding region of five strains of peanut stripe virus (PStV) from Thailand have been cloned and sequenced. Phylogenetic comparisons of these strains, known as T1, T3, T5, T6 and T7, and related sequences showed that these strains are indeed strains of PStV. Further, PStV strains appear to be related to each other according to their geographic origin. That is, the Thai strains are more closely related to each other than they are to strains from the USA or Indonesia, despite the variety of symptoms caused by these strains and the overlap of symptom types between the strains from different locations. Like other PStV strains, PStV-Thai can be considered strains of bean common mosaic virus (BCMV) but can be distinguished from bean-infecting strains of BCMV and blackeye cowpea mosaic virus (BlCMV) through sequence and host range. No evidence was found that PStV-Thai strains, unlike PStV-Ib, are recombinants of PStV and BlCMV, although the T3 strain may be a recombinant of different PStV sequences. Phylogenetic analyses of viruses of the BCMV group suggest that acquisition of the ability to infect peanut may have occurred only once.

First complete genome sequence of a capsicum chlorosis tospovirus isolate from Australia with an unusually large S RNA intergenic region

The first complete genome sequence of capsicum chlorosis virus (CaCV) from Australia was determined using a combination of Illumina HiSeq RNA and Sanger sequencing technologies. Australian CaCV had a tripartite genome structure like other CaCV isolates. The large (L) RNA was 8913 nucleotides (nt) in length and contained a single open reading frame (ORF) of 8634 nt encoding a predicted RNA-dependent RNA polymerase (RdRp) in the viral-complementary (vc) sense. The medium (M) and small (S) RNA segments were 4846 and 3944 nt in length, respectively, each containing two non-overlapping ORFs in ambisense orientation, separated by intergenic regions (IGR). The M segment contained ORFs encoding the predicted non-structural movement protein (NSm; 927 nt) and precursor of glycoproteins (GP; 3366 nt) in the viral sense (v) and vc strand, respectively, separated by a 449-nt IGR. The S segment coded for the predicted nucleocapsid (N) protein (828 nt) and non-structural suppressor of silencing protein (NSs; 1320 nt) in the vc and v strand, respectively. The S RNA contained an IGR of 1663 nt, being the largest IGR of all CaCV isolates sequenced so far. Comparison of the Australian CaCV genome with complete CaCV genome sequences from other geographic regions showed highest sequence identity with a Taiwanese isolate. Genome sequence comparisons and phylogeny of all available CaCV isolates provided evidence for at least two highly diverged groups of CaCV isolates that may warrant re-classification of AIT-Thailand and CP-China isolates as unique tospoviruses, separate from CaCV.

Differentiation of Peanut Seedborne Potyviruses and Cucumoviruses by RT-PCR

Seedborne peanut viruses pose important constraints to peanut production and safe movement of germ plasm. They also pose a risk of accidental introduction into previously disease-free regions. We have developed reverse transcription-polymerase chain reaction (RT-PCR) assays based on identical cycling parameters which identified peanut stripe, Peanut mottle, Peanut stunt, and Cucumber mosaic viruses through production of specific DNA fragments of 234 bp, 327 bp, 390 bp, and 133 bp, respectively. Assay sensitivity in the picogram range was achieved. The two potyviruses and two cucumoviruses could be differentiated using duplex RT-PCR assays. These assays should be useful for testing peanut leaves or seeds for virus identification in epidemiological studies, seed testing or in post-entry quarantine.

Ankylosing spondylitis and HLA-B27: restriction fragment length polymorphism and sequencing of an HLA-B27 allele from a patient with ankylosing spondylitis.

Two groups of patients with ankylosing spondylitis (AS) from England and Poland were examined for restriction fragment length polymorphisms (RFLPs) associated with the disease. No preferential association was found between the 9.2 kb PvuII fragment in HLA-B27 positive patients with AS compared with HLA-B27 healthy subjects as had been previously reported. In the English group, however, a 14 kb PvuII fragment was more common in HLA-B27 positive subjects with AS than in normal controls. Also 4.6 and 3.7 kb PvuII fragments were more prevalent in subjects without AS than in the group with AS, but these results were confined to the English group. Furthermore, the sequence of an HLA-B*2705 gene isolated from a patient with AS was examined, and no significant differences were found compared with the sequence isolated from a healthy subject. There do not seem to be significant genetic differences in the coding or in the regulatory region in HLA-B27 alleles, in subjects with or without AS.

Genetic signatures indicate widespread antibiotic resistance and phage infection in microbial communities of the McMurdo Dry Valleys, East Antarctica

The McMurdo Dry Valleys of Antarctica support extensive yet cryptic microbial communities but little evidence for ‘top-down’ herbivory control. A question therefore arises as to how standing microbial biomass is regulated. Here, we present results from a survey of soil and rock microbial community metagenomes using the GeoChip microarray that demonstrate antibiotic resistance and phage infection are widespread. We interrogated a range of dry valley locations from maritime to extreme inland sites. Antibiotic resistance genes were identified in three categories: beta-lactamases, tetracycline and vanomycin plus a range of transporter genes. Frequency of recovery generally reflected microbial diversity, with greatest abundance among Halobacteria, Proteobacteria and the photosynthetic bacteria (Chlorobi, Chloroflexi and Cyanobacteria). However, no clear differences between locations and soil/rock communities were apparent. Phage signals were also recovered from all locations in soil and rock communities. The Leviviridae, Myoviridae, Podoviridae and Siphoviridae were ubiquitous . The Corticoviridae occurred only in moisture-sufficient hyporheic soils, the Microviridae occurred only in maritime and hyporheic sites and an unidentified group within the order Caudovirales occurred only at dry inland sites. We postulate that widespread antibiotic resistance indicates potential inter-specific interaction and that phage signals indicate possible ‘bottom-up’ trophic regulation in the dry valleys.

PCR rescue and analysis of transgene sequences directly from crude extracts of transgenic embryos and plants

We report the application of a PCR-based method in conjunction with automated sequencing for the reliable detection and verification of transgenes in crude extracts of leaf and callus tissue from different plant species. Transformed tissue can be identified easily at any stage of the regeneration process, whether it is via embryogenesis or organogenesis. This allows researchers to focus their attention and resources on truly transformed tissues and avoid unwittingly culturing untransformed tissues. This protocol can also be used to rescue relatively large PCR products as well as duplexing the detection of transgenes. Direct sequencing of the PCR products allows confirmation of the integrity of the transgenein planta.