Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20–40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

Angiosperms form the largest phylum within the Plantae kingdom and show remarkable genetic variation due to the considerable difference in the nuclear genome size of each species. Transposable elements (TEs), mobile DNA sequences that can amplify and change their chromosome position, account for much of the difference in nuclear genome size between individual angiosperm species. Considering the dramatic consequences of TE movement, including the complete loss of gene function, it is unsurprising that the angiosperms have developed elegant molecular strategies to control TE amplification and movement. Specifically, the RNA-directed DNA methylation (RdDM) pathway, directed by the repeat-associated small-interfering RNA (rasiRNA) class of small regulatory RNA, forms the primary line of defense to control TE activity in the angiosperms. However, the miniature inverted-repeat transposable element (MITE) species of TE has at times avoided the repressive effects imposed by the rasiRNA-directed RdDM pathway. MITE proliferation in angiosperm nuclear genomes is due to their preference to transpose within gene-rich regions, a pattern of transposition that has enabled MITEs to gain further transcriptional activity. The sequence-based properties of a MITE results in the synthesis of a noncoding RNA (ncRNA), which, after transcription, folds to form a structure that closely resembles those of the precursor transcripts of the microRNA (miRNA) class of small regulatory RNA. This shared folding structure results in a MITE-derived miRNA being processed from the MITE-transcribed ncRNA, and post-maturation, the MITE-derived miRNA can be used by the core protein machinery of the miRNA pathway to regulate the expression of protein-coding genes that harbor homologous MITE insertions. Here, we outline the considerable contribution that the MITE species of TE have made to expanding the miRNA repertoire of the angiosperms.

TheDEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1)gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1’s primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least twoDEMgenes.Arabidopsishas twoDEMgenes,DEM1andDEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygousdem1 dem2double mutants were not recovered, and plants carrying a single functionalDEM1allele and no functional copies ofDEM2, i.e.DEM1/dem1 dem2/dem2plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast,dem1/dem1 DEM2/dem2plants showed normal levels of fertility, indicating thatDEM2plays a more important role thanDEM1in gamete viability. The increased importance ofDEM2in gamete viability correlated with higher mRNA levels ofDEM2compared toDEM1in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functionalDEMalleles inherited following meiosis, but also on the number of functionalDEMalleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Most flowering plant species contain at least two copies of the DEFECTIVE EMBRYO AND MERISTEMS (DEM) gene with the encoded DEM proteins lacking homology to proteins of known biochemical function. In tomato (Sl; Solanum lycopersicum), stable mutations in the SlDEM1 locus result in shoot and root meristem defects with the dem1 mutant failing to progress past the cotyledon stage of seedling development. Generation of a Somatic Mutagenesis of DEM1 (SMD) transformant line in tomato allowed for the characterization of SlDEM1 gene function past the seedling stage of vegetative development with SMD plants displaying a range of leaf development abnormalities. Further, the sectored or stable in planta expression of specific regions of the SlDEM1 coding sequence also resulted in the generation of tomato transformants that displayed a range of vegetative development defects, which when considered together with the dem1 mutant seedling and SMD transformant line phenotypic data, allowed for the assignment of SlDEM1 gene function to early embryo development, adaxial epidermis cell development, lateral leaf blade expansion, and mesophyll cell proliferation and differentiation.

Background: Rapid and objective assessment of foliar disease is important for screening host responses and evaluating novel control agents. We developed a complementary suite of low-cost methods for quantitative assessment of wheat leaf rust, combining an ImageJ-based disease quantification workflow (RONUM) with detached-leaf and soil-free whole-plant infection assays. Results: RONUM enabled semi-automated quantification of disease metrics following user-guided calibration. Against manually corrected reference image sets from whole plant leaf scans and detached leaves, disease estimates showed strong agreement with reference measurements and were reproducible following independent calibration by multiple users, and by pixel agreement optimisation to the reference masks. Benchmarked against another published ImageJ workflow, RONUM also showed closer agreement with manually corrected measurements, particularly for images containing chlorosis, necrosis or poor leaf health. Post-processing options were quantitatively assessed, showing little influence over total pustule-area estimates but substantial effects upon pustule count estimates. Two complementary infection assays were presented to demonstrate example applications of the quantification workflow: a soil-free whole-plant assay for non-destructive tracking of leaf rust development over an 18-day infection time course; and a benchtop-scale detached-leaf assay. The detached leaf assay provided reproducible disease quantification across 13 wheat lines with contrasting disease phenotypes. Exploratory quantification of chlorosis: pustule area ratios substantially separated the susceptible lines from those harbouring pathotype-specific Lr resistance, showcasing a potential application for collecting biologically relevant phenotype data typically unavailable to image-based quantitation. Finally, a modified detached leaf assay with nylon-mesh treatment reservoir was developed and assessed by establishing a dose-response curve to a propiconazole fungicide formulation, with an estimated IC₅₀ of 20.49 ppm formulation (95% CI 14.64–28.11 ppm). Conclusions: These methods provide accessible tools for quantitative wheat rust research using standard laboratory equipment and freely available software. The principal value of RONUM lies in flexible calibration to individual experimental systems followed by rapid and consistent quantification across replicate datasets, along with requisite annotation and output summaries for streamlined record keeping. Combined with complementary whole-plant and detached-leaf assays, the workflow enables quantitative assessment of disease progression, and the screening of candidate treatments or putative traits. While further validation is required, the preliminary application of these tools highlights potential application toward image-based characterisation of agronomically important traits.