Identification of Potential Host Plants of Sap-Sucking Insects (Hemiptera: Cicadellidae) Using Anchored Hybrid By-Catch Data

Reliable host plant records are available for only a small fraction of herbivorous insect species, despite their potential agricultural importance. Most available data on insect–plant associations have been obtained through field observations of occurrences of insects on particular plants. Molecular methods have more recently been used to identify potential host plants using DNA extracted from insects, but most prior studies using these methods have focused on chewing insects that ingest tissues expected to contain large quantities of plant DNA. Screening of Illumina data obtained from sap feeders of the hemipteran family Cicadellidae (leafhoppers) using anchored hybrid enrichment indicates that, despite feeding on plant fluids, these insects often contain detectable quantities of plant DNA. Although inclusion of probes for bacterial 16S in the original anchored hybrid probe kit yielded relatively high detection rates for chloroplast 16S, the Illumina short reads also, in some cases, included DNA for various plant barcode genes as “by-catch”. Detection rates were generally only slightly higher for Typhlocybinae, which feed preferentially on parenchyma cell contents, compared to other groups of leafhoppers that feed preferentially on phloem or xylem. These results indicate that next-generation sequencing provides a powerful tool to investigate the specific association between individual insect and plant species.

Plant DNA Repair and Agrobacterium T−DNA Integration

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T−DNA integration into the plant genome.

Accurate Direct PCR with Arabidopsis and rice

Methods for PCR that avoid costly and time consuming plant DNA purification have not been widely adopted, partly because their efficacy is unclear. Here, we compare different sampling methods for Direct PCR in Arabidopsis and rice. CutTip, stabbing a pipette tip into a plant organ and depositing the tip of the pipette tip into the reaction buffer, yielded high accuracy for genotyping and detection of CRISPR-induced mutations. This did not require visible tissue fragments in the reactions. We demonstrate the usefulness of this method in sampling many locations within a single plant to identify a rare CRISPR-mutated sector. These methods are simple, inexpensive and can help address the challenge of genotyping and genome editing at different scales with high accuracy. The methods also simplify the application of PCR in the field.

Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles

Junaid M, Purwantara A, Guest G. 2021. Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles. Biodiversitas 22: 2838-2843. Understanding the biology of the fastidious Basidiomycete Ceratobasidium theobromae occupying host-tissue is essential for plant disease management. Direct pathogen DNA extraction from infected plant tissue avoids the need for isolation in artificial media. We report a modified DNA isolation protocol to obtain total plant DNA designed to overcome DNA extraction and isolation problems caused by infected petioles rich in polysaccharides and phenolic substances as a primary source of gummosis. This study examined and compared total plant DNA isolated from petioles high in polysaccharides and polyphenolic compounds using two methods: conventional CTAB lysis buffer and Kits (the standard method), and a new modified CTAB protocol to address these PCR inhibitors. The modified method resulted in higher quality and quantity of C. theobromae crude DNA and amplified PCR product. The modified method produced large quantities of clear, transparent, aqueous DNA-containing lysate (crude DNA) with a clear separation between the upper crude DNA and organic waste layers. Mean DNA absorbance was 1.80, and the lowest DNA yield was 836.6 ng/µl. With the standard method, the blurred, viscous lysate obtained showed signs of gummosis, with poor separation between layers of crude DNA, polysaccharides, protein, and organic waste layers and low yield. Gel electrophoresis indicated poor quality DNA extract. We conclude that this modified method will be valuable for genetic diversity and disease studies in a range of previously challenging plant tissues and their pathogens.

Plant DNA polymerases α and δ mediate replication of geminiviruses

Geminiviruses are causal agents of devastating diseases in crops. Geminiviruses have circular single-stranded (ss) DNA genomes that are replicated in the nucleus of the infected plant cell through double-stranded (ds) DNA intermediates by the plant DNA replication machinery. Which host DNA polymerase mediates geminiviral multiplication, however, has so far remained elusive. Here, we show that subunits of the nuclear replicative DNA polymerases α and δ physically interact with the geminivirus-encoded replication enhancer protein, C3, and that these polymerases are required for viral replication. Our results suggest that, while DNA polymerase α is essential to generate the viral dsDNA intermediate, DNA polymerase δ mediates the synthesis of new copies of the geminiviral ssDNA genome, and that the virus-encoded C3 may act selectively, recruiting DNA polymerase δ over ε to favour productive replication.