Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function.

The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis.

Mapping, complementation, and targets of the cysteine protease actinidin in kiwifruit.

Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.

Changes in Leaf-Level Nitrogen Partitioning and Mesophyll Conductance Deliver Increased Photosynthesis for Lolium perenne Leaves Engineered to Accumulate Lipid Carbon Sinks.

Diacylglycerol acyl-transferase (DGAT) and cysteine oleosin (CO) expression confers a novel carbon sink (of encapsulated lipid droplets) in leaves of Lolium perenne and has been shown to increase photosynthesis and biomass. However, the physiological mechanism by which DGAT + CO increases photosynthesis remains unresolved. To evaluate the relationship between sink strength and photosynthesis, we examined fatty acids (FA), water-soluble carbohydrates (WSC), gas exchange parameters and leaf nitrogen for multiple DGAT + CO lines varying in transgene accumulation. To identify the physiological traits which deliver increased photosynthesis, we assessed two important determinants of photosynthetic efficiency, CO2 conductance from atmosphere to chloroplast, and nitrogen partitioning between different photosynthetic and non-photosynthetic pools. We found that DGAT + CO accumulation increased FA at the expense of WSC in leaves of L. perenne and for those lines with a significant reduction in WSC, we also observed an increase in photosynthesis and photosynthetic nitrogen use efficiency. DGAT + CO L. perenne displayed no change in rubisco content or Vcmax but did exhibit a significant increase in specific leaf area (SLA), stomatal and mesophyll conductance, and leaf nitrogen allocated to photosynthetic electron transport. Collectively, we showed that increased carbon demand via DGAT+CO lipid sink accumulation can induce leaf-level changes in L. perenne which deliver increased rates of photosynthesis and growth. Carbon sinks engineered within photosynthetic cells provide a promising new strategy for increasing photosynthesis and crop productivity.

Genetic transformation of western clover (Trifolium occidentale D. E. Coombe.) as a model for functional genomics and transgene introgression in clonal pasture legume species.

BACKGROUND: Western clover (Trifolium occidentale) is a perennial herb with characteristics compatible for its development as an attractive model species for genomics studies relating to the forage legume, white clover (Trifolium repens). Its characteristics such as a small diploid genome, self-fertility and ancestral contribution of one of the genomes of T. repens, facilitates its use as a model for genetic analysis of plants transformed with legume or novel genes. RESULTS: In this study, a reproducible transformation protocol was established following screening of T. occidentale accessions originating from England, Ireland, France, Spain and Portugal. The protocol is based upon infection of cotyledonary explants dissected from mature seed with the Agrobacterium tumefaciens strain GV3101 carrying vectors which contain the bar selection marker gene. Transformation frequencies of up to 7.5% were achieved in 9 of the 17 accessions tested. Transformed plants were verified by PCR and expression of the gusA reporter gene, while integration of the T-DNA was confirmed by Southern blot hybridisation and segregation of progeny in the T1 generation. CONCLUSIONS: Development of this protocol provides a valuable contribution toward establishing T. occidentale as a model species for white clover. This presents opportunities for further improvement in white clover through the application of biotechnology.