Callus Culture as a Prospective Biosensor: Examining Fusarium graminearum Infection and Exploring the Use of RUBY as a Pathogen-Inducible Reporter

Abstract

With climate change and globalization contributing to the spread of plant diseases and a reliance on visual symptoms for detection, agriculturalists face continual challenges in identifying the presence of pathogens with enough lead time for effective mitigation, highlighting a need for novel early plant pathogen detection technologies. This thesis introduces an unconventional approach for rapid plant pathogen detection: transgenic callus culture. Callus is comprised of masses of undifferentiated cells, appealing for this application due to its potential to serve as a compact model of whole plants, including response to threats of plant pathogens. When transformed with a paired pathogen-inducible promoter (i.e., switches on in the presence of pathogens) and reporter (i.e., genes that trigger a phenotypic alteration when activated), this tissue could be a prospective candidate for a plant biosensor (i.e., phytosensor). We sought to determine if callus of Arabidopsis thaliana could become infected and tested this with the fungal plant pathogen, Fusarium graminearum. To investigate this, we assessed qualitative and quantitative markers of early infection in inoculated whole plants and callus, using a visual Fusarium-Arabidopsis Disease (FAD) rating system and quantifying deoxynivalenol (DON) (a mycotoxin) and ergosterol (i.e., an indicator of fungal biomass). We compared measurements across three accessions of A. thaliana with a range of known disease susceptibilities at 7, 14, and 21 days post-inoculation (DPI). All inoculated plants exhibited signs and symptoms of fungal infection such as mycelial growth, drying, and constriction, and the A. thaliana accessions varied significantly in FAD ratings (P=0.0329 for accession, P=0.0483 for timepoint, and P=0.0025 for experiment). DON was detected in flower and seed samples from infected A. thaliana plants. Inoculated calli exhibited infection symptoms, and DON was measured in some but not all callus samples. Ergosterol was detected in all inoculated callus samples, and an analysis of variance revealed significant differences in concentration means (P=0.0163 for accession, P<0.0001 for timepoint, and P=0.0182 for experiment). Another aspect of this research focused on genetically combining the pathogenesis-related protein 1 (PR-1) promoter gene from A. thaliana, which is activated in response to pathogen presence, with a RUBY reporting system that, when induced, turns tissue a red color, perceptible to the unaided human eye. This system was molecularly synthesized and transformed into the genome of A. thaliana plants, the seeds of which can be used to grow phytosensor plants or callus in the future. Preliminary testing of T1 plants using flg22 leaf infiltration to trigger the pathogen-inducible system has produced inconsistent yet promising results, with at least one plant from each transformed accession exhibiting RUBY expression. Further investigation into the pathogen-response capabilities of this system can confirm the efficacy of using reporter callus in phytopathogen detection. The work presented here can inform the design of novel phytosensors, hopefully contributing to the development of portable, on-site biosensor technology that can immediately alert the presence of agronomically important pathogens and equip growers with a potential early warning system for diseases that threaten their crops.