D. Barry Scott

Surrogate transformation of perennial ryegrass,Lolium perenne, using genetically modifiedAcremonium endophyte

SummaryConditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/μg DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The β-glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.

Molecular systematics and evolution in New Zealand: applications to cryptic skink species

Abstract The polymerase chain reaction (PCR) and DNA sequencing are being used for the study of the origin and evolution of New Zealand plants and animals. Here we describe methods for rapidly obtaining DNA sequences from small amounts of fresh and preserved tissue, and apply them to the problem ofresolving relationships among skinks of the Austrnlasian genus Leiolopisma. DNA extracted from frozen tail muscle was used for the main part of the study. Part ofthe mitochondrial12S ribosomal RNA gene was isolated using PCR. Direct sequencing of this 400 nucleotide region supports the separation of “L. nigriplantare” into several genetically distinct but morphologically similar species. The sequence data have also identified a potential case of hybridisation between two sympatric species in Otago, L. nigriplantare polychroma and L. maccanni. The data set supports the view that skinks have been in New Zealand very much longer than five million years. From these results we expect that application of the PCR and o...

Molecular cloning of a nodulation gene from fast- and slow-growing strains of Lotus rhizobia

SummaryCosmids containing a nodulation gene from Rhizobium loti NZP2037 were isolated using a 12.8 kb nod:: Tn5EcoRI fragment from the Nod- mutant strain PN233, as a hybridisation probe. A physical map of the nod region was established using the enzymes EcoRI and HindIII and the site of insertion of Tn5 in PN233 determined. Site-specific exchange of the cloned nod:: Tn5 fragment demonstrated that Tn5, and not an indigenous insertion sequence, was responsible for the nod mutation in PN233. The nod cosmids isolated complemented the Nod- phenotype of strain PN233 but restoration of the Fix phenotype was variable suggesting a need for marker rescue to occur before nitrogen fixation occurred.Corresponding nod cosmids were isolated from a R. loti strain, NZP2213, that forms ineffective tumour-like structures on Lotus pedunculatus and from the slow-growing strain (Bradyrhizobium sp), CC814s, by in planta complementation of PN233. Hybridisation experiments suggested that the nod gene region of R. loti NZP2037 was more homologous to Bradyrhizobium strain CC814s than with a nod gene region of R. trifolii strain PN100. However, transfer of the R. trifolii nod cosmid into the R. loti Nod mutant PN233, restored the ability of this strain to initiate nodules on Lotus pedunculatus.

Exploring molecular signaling in plant-fungal symbioses using high throughput RNA sequencing

Plant-fungal symbioses are a common feature in nature. They vary from pathogenic interactions, where fungi subvert plant resources for their own use, to mutualistic associations, where both fungus and host benefit from the interaction. Although the ecological importance of plant-fungal symbioses has long been recognized and the biology of several key associations are now well studied, new technologies have the potential to allow fresh insight into the molecular basis of plant-fungal interactions. One such technique – high throughput RNA sequencing – has recently been used to explore the molecular basis of cross-species communications. Here, we give a brief overview of this emerging technology, and present a general guide for employing the methodology to dissect plant-fungal symbiosis.

Isoprenoids: Gene Clusters and Chemical Puzzles

The isoprenoids are one of the most chemically diverse groups of natural products found in nature, with greater than 23,000 compounds identified (Dewick, 2002). All are derived from linear isoprenoid diphosphates synthesized from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by a family of prenyltransferases that catalyze the sequential condensations of IPP with allylic isoprenoid diphosphates (Figure 8.1). The primary isoprenoid substrates can be derived from either the mevalonate pathway or the more recently discovered deoxyxylulose phosphate (mevalonate-independent) pathway (Rohmer et al., 1993), that is found in eubacteria and plants (Dewick, 2002). Most fungi contain both farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) synthases, which catalyze the formation of the linear C15 and C20 isoprenoids, FPP and GGPP, respectively. FPP is a key branch point in the isoprene pathway from where additional prenyl transferases catalyze the synthesis of precursors for sterols, ubiquinones, dolichols, sesquiterpenes, and farnesylated proteins. GGPP is the substrate for another group of prenyl transferases involved in the biosynthesis of gibberellins, carotenoids, indole-diterpenes, and geranylgeranylated proteins (Figure 8.1).

Structure of rhizolotine, a novel opine-like metabolite from Lotus tenuis nodules

A novel opine-like metabolite rhizolotine (1) has been isolated from an ethanol extract of root nodules from Lotus tenuis inoculated with Rhizobium loti NZP2037; the structure and conformation are reported.

Epichloë hybrida, sp. nov., an emerging model system for investigating fungal allopolyploidy

ABSTRACT Endophytes of the genus Epichloë (Clavicipitaceae, Ascomycota) frequently occur within cool-season grasses and form interactions with their hosts that range from mutualistic to antagonistic. Many Epichloë species have arisen via interspecific hybridization, resulting in species with two or three subgenomes that retain all or nearly all of their original parental genomes, a process termed allopolyploidization. Here, we characterize Epichloë hybrida, sp. nov., a mutualistic species that has increasingly become a model system for investigating allopolyploidy in fungi. The Epichloë species so far identified as the closest known relatives of the two progenitors of E. hybrida are E. festucae var. lolii and E. typhina. We confirm that the nuclear genome of E. hybrida contains two homeologs of most protein-coding genes from E. festucae and E. typhina, with genome-wide gene expression analysis indicating a slight bias in overall gene expression from the E. typhina subgenome. Mitochondrial DNA is detectable only from E. festucae, whereas ribosomal DNA is detectable only from E. typhina. Inheriting ribosomal DNA from just one parent might be expected to preferentially favor interactions with ribosomal proteins from the same parent, but we find that ribosomal protein genes from both parental subgenomes are nearly all expressed equally in E. hybrida. Finally, we provide a comprehensive set of resources for this model system that are intended to facilitate further study of fungal hybridization by other researchers.