Multiplex qPCR assays to distinguish individual species of arbuscular mycorrhizal fungi from roots and soil.

Currently, root colonization measurements of arbuscular mycorrhizal fungi (AMF) require staining and microscopy, and species-level identification of the fungi by such observations is not possible. Here, we present novel multiplex real-time PCR assays targeting the glomalin genes of 11 different species of AMF commonly found in temperate agricultural soils, which independently detect and measure the abundance of these fungi using DNA extracts from soil and or root tissue. The availability of these tools will not only increase throughput in determining levels of root colonization but can provide species-specific levels of root colonization from a single sample. This will help to establish which AMF species, or combinations of different species, provide the most benefits to crops, and will aid in the development of AMF for use as biofertilizers.

Real-Time PCR Assays to Detect and Distinguish the Rapid 'Ohi'a Death Pathogens Ceratocystis lukuohia and C. huliohia.

Ceratocystis lukuohia and C. huliohia are recently described fungal species that cause rapid 'ohi'a death (ROD) of Metrosideros polymorpha, Hawaii's most abundant and ecologically important native species. Although the pathogens are now widespread on Hawai'i Island, a major effort is underway to study and manage affected forests, and particularly to prevent the disease from spreading to other islands in the State or throughout the Pacific. Rapid and accurate detection is critical. Molecular diagnostic real-time PCR protocols were developed to detect and distinguish the two pathogens, suitable for detection of fungal DNA from extracts of wood, soil, and insect frass. The assays detect as few as 2 to 4 or 16 spores of C. huliohia or C. lukuohia, respectively. These assays are valuable tools for monitoring disease spread and offer a significant advantage over culture-based methods for diagnostics, requiring <1 day to arrive at definitive results.

RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing.

Transcripts of plant organelle genes are modified by cytidine-to-uridine (C-to-U) RNA editing, often changing the encoded amino acid predicted from the DNA sequence. Members of the PLS subclass of the pentatricopeptide repeat (PPR) motif-containing family are site-specific recognition factors for either chloroplast or mitochondrial C targets of editing. However, other than PPR proteins and the cis-elements on the organelle transcripts, no other components of the editing machinery in either organelle have previously been identified. The Arabidopsis chloroplast PPR protein Required for AccD RNA Editing 1 (RARE1) specifies editing of a C in the accD transcript. RARE1 was detected in a complex of >200 kDa. We immunoprecipitated epitope-tagged RARE1, and tandem MS/MS analysis identified a protein of unknown function lacking PPR motifs; we named it RNA-editing factor interacting protein 1 (RIP1). Yeast two-hybrid analysis confirmed RIP1 interaction with RARE1, and RIP1-GFP fusions were found in both chloroplasts and mitochondria. Editing assays for all 34 known Arabidopsis chloroplast targets in a rip1 mutant revealed altered efficiency of 14 editing events. In mitochondria, 266 editing events were found to have reduced efficiency, with major loss of editing at 108 C targets. Virus-induced gene silencing of RIP1 confirmed the altered editing efficiency. Transient introduction of a WT RIP1 allele into rip1 improved the defective RNA editing. The presence of RIP1 in a protein complex along with chloroplast editing factor RARE1 indicates that RIP1 is an important component of the RNA editing apparatus that acts on many chloroplast and mitochondrial C targets.

Inoculation of black turtle beans (Phaseolus vulgaris) with mycorrhizal fungi increases the nutritional quality of seeds.

The use of arbuscular mycorrhizal fungi (AMF) as biofertilizers has proven successful in boosting the yield and nutritional quality of a variety of crops. AMF associate with plant roots and exchange soil nutrients for photosynthetically derived C in the form of sugars and lipids. Past research has shown that not all AMF species are equal in their benefit to nutrient uptake and crop health, and that the most beneficial AMF species appear to vary by host species. Although an important human food staple, especially in developing regions where nutrient deficiency is a prevalent threat to public health, little work has been done to test the effectiveness of AMF in enhancing the nutritional quality of common bean (Phaseolus vulgaris L.). Therefore, our objective was to determine the most beneficial AMF species for inoculation of this important crop. We inoculated black beans (Phaseolus vulgaris black turtle beans) with eight individual AMF species and one mixed species inoculum in an outdoor pot trial over 3 months and assessed the extent to which they altered yield, mineral nutrient and anthocyanin concentration of seeds and leaf tissues. Despite seeing no yield effects from inoculation, we found that across treatments percent root length colonized by AMF was positively correlated with plant tissue P, Cu, and Zn concentration. Underlying these broad benefits, seeds from plants inoculated with three AMF species, Claroideoglomus claroideum (+15%), Funneliformis mosseae (+13%), and Gigaspora rosea (+11%) had higher P concentration than non-mycorrhizal plants. C. claroideum also increased seed potassium (K) and copper (Cu), as well as leaf aluminum (Al) concentration making it a promising candidate to further test the benefit of individual AMF species on black bean growth in field trials.

A comparative genomics approach identifies a PPR-DYW protein that is essential for C-to-U editing of the Arabidopsis chloroplast accD transcript.

Several nuclear-encoded proteins containing pentatricopeptide repeat (PPR) motifs have previously been identified to be trans-factors essential for particular chloroplast RNA editing events through analysis of mutants affected in chloroplast biogenesis or function. Other PPR genes are known to encode proteins involved in other aspects of organelle RNA metabolism. A function has not been assigned to most members of the large plant PPR gene family. Arabidopsis and rice each contain over 400 PPR genes, of which about a fifth exhibit a C-terminal DYW domain. We describe here a comparative genomics approach that will facilitate identification of the role of RNA-binding proteins in organelle RNA metabolism. We have implemented this strategy to identify an Arabidopsis nuclear-encoded gene RARE1 that is required for editing of the chloroplast accD transcript. RARE1 carries 15 PPR motifs, an E/E+ and a DYW domain, whereas previously reported editing factors CRR4, CRR21, and CLB19 lack a DYW domain. The accD gene encodes the beta carboxyltransferase subunit of acetyl coA carboxylase, which catalyzes the first step in fatty acid biosynthesis in chloroplasts. Despite a lack of accD C794 editing and lack of restoration of an evolutionarily conserved leucine residue in the beta carboxyltransferase protein, rare1 mutants are unexpectedly robust and reproduce under growth room conditions. Previously the serine-to-leucine alteration caused by editing was deemed essential in the light of the finding that a recombinantly expressed "unedited" form of the pea acetyl coA carboxylase was catalytically inactive.

A qPCR-based method for detecting parasitism of Fopius arisanus (Sonan) in oriental fruit flies, Bactrocera dorsalis (Hendel).

BACKGROUND: Parasitism detection and species identification are necessary in fruit fly biological control. Currently, release of mass-reared Fopius arisanus is practiced worldwide, as it is effective in controlling Bactrocera dorsalis and Ceratitis capitata. To detect and assess parasitism in parasitoid mass-rearing colonies and parasitism levels in field populations across all life stages of hosts, the development of a rapid, specific and sensitive method is important. RESULTS: A species-specific probe was designed for F. arisanus, as well as a universal tephritid probe. Utilizing rapid DNA extraction techniques coupled with quantitative-PCR, a simple and fast assay has been developed to detect parasitism of F. arisanus that is sensitive enough to detect the parasitoid across all developmental stages, including a single egg per host egg or 0.25 ng of parasitoid DNA in 40 ng of host DNA. The qPCR methods also detect a higher parasitism rate when compared with rearing-based methods where parasitism rate is based on wasp emergence and where unemerged wasps are not included. CONCLUSION: This method is a rapid, sensitive and specific technique to determine the parasitism rate of F. arisanus across all life stages of B. dorsalis, which will be useful to predict parasitoid output from mass rearing and evaluate the outcome of pest suppression after mass release in the field.

Cross-competition in editing of chloroplast RNA transcripts in vitro implicates sharing of trans-factors between different C targets.

C-->U plant organellar RNA editing is required for the translation of evolutionarily conserved and functional proteins. 28 different C targets of RNA editing have been identified in maize chloroplasts, and hundreds of Cs are edited in mitochondria. Mutant analysis in Arabidopsis has indicated that absence of a single site-specific recognition protein can result in loss of editing of a single C target, raising the possibility that each C target requires a recognition protein. Here we show that transcripts encompassing two editing sites, ZMrpoB C467 and ZMrps14 C80, can compete editing activity from each other in vitro despite limited sequence similarity. The signal causing competition overlaps a 5'-cis element required for editing efficiency. A single five-nucleotide mutation spanning the region from -20 to -16 relative to the edited C of rpoB C467 is sufficient to eliminate its substrate editing as well as its ability to compete editing activity from rps14 C80 substrates. A corresponding mutation in an rps14 C80 competitor likewise eliminated its ability to compete editing activity from rpoB C467 substrates. Taken together, our results indicate that the RNA sequences mediating both editing efficiency and cross-competition are highly similar and that a common protein is involved in their editing. Sharing of trans-factors can facilitate editing of the large number of different C targets in plant organelles so that a different protein factor would not be required for every editing site.