Developmentally regulated activation of defense allows for rapid inhibition of infection in age-related resistance to Phytophthora capsici in cucumber fruit

Abstract

Background Age-related resistance (ARR) is a developmentally regulated phenomenon conferring resistance to pathogens or pests. Although ARR has been observed in several host-pathogen systems, the underlying mechanisms are largely uncharacterized. In cucumber, rapidly growing fruit are highly susceptible to Phytophthora capsici but become resistant as they complete exponential growth. We previously demonstrated that ARR is associated with the fruit peel and identified gene expression and metabolomic changes potentially functioning as preformed defenses. Results Here, we compare the response to infection in fruit at resistant and susceptible ages using microscopy, quantitative bioassays, and weighted gene co-expression analyses. We observed strong transcriptional changes unique to resistant aged fruit 2–4 h post inoculation (hpi). Microscopy and bioassays confirmed this early response, with evidence of pathogen death and infection failure as early as 4 hpi and cessation of pathogen growth by 8–10 hpi. Expression analyses identified candidate genes involved in conferring the rapid response including those encoding transcription factors, hormone signaling pathways, and defenses such as reactive oxygen species metabolism and phenylpropanoid biosynthesis. Conclusion The early pathogen death and rapid defense response in resistant-aged fruit provide insight into potential mechanisms for ARR, implicating both pre-formed biochemical defenses and developmentally regulated capacity for pathogen recognition as key factors shaping age-related resistance.

Quantitative High-Throughput, Real-Time Bioassay for Plant Pathogen Growth in vivo

“…Similar results were observed in all three experiments with growth largely ceasing in the 16 dpp “Poinsett 76” fruit by 15 hpi in each case. These results are consistent with electron microscopy showing evidence of pathogen death on 16 dpp “Poinsett 76” fruit by 8 hpi ( Mansfeld et al, 2020 ).…”

“…For the ARR experiments, hand-pollinated fruits were produced on plants in the greenhouse under growing conditions as described by Ando et al (2015) . Fruits of “Poinsett 76,” which exhibits ARR ( Mansfeld et al, 2020 ) were harvested at 8 vs. 16 days post pollination (dpp); 16 dpp fruit were used for comparisons of “Poinsett 76” (ARR+) and “Gy14” (ARRı-). For the comparisons of young fruit from “Gy14” and “A4-3,” trellised plants were grown in the field and pollinated by bees under growing conditions as described by Grumet and Colle (2017) .…”

“…Zoospore suspensions were prepared from P. capsici isolate NY0664-1 expressing RFP gene tdTomato , kindly provided by C. Smart, Cornell University ( Dunn et al, 2013 ). This isolate was originally obtained from pepper as described by Dunn et al (2013) and also has been used for previous experiments with cucumber (e.g., Mansfeld et al, 2020 ). The pathogen was cultured on V8 agar medium as described by Mansfeld et al (2017) .…”

“…Harvested cucumber fruits and pepper leaves were washed and surface sterilized as previously described ( Colle et al, 2014 ). Young cucumber fruit were inoculated with two, 30 μL droplets of 1 × 10 4 zoospores/mL of P. capsici isolate NY 0664-1-RFP (as per Colle et al, 2014 ); fruits for ARR experiments were inoculated with two, 30 μl droplets of 1 × 10 5 or 5 × 10 5 zoospores/ml (as per Mansfeld et al, 2020 ). Fruit were then incubated for 5 days at room temperature (22 C) in covered plastic trays lined with wet paper towels, to maintain high humidity as described by Colle et al (2014) .…”

“…This approach has been used in numerous fungal, oomycete and bacterial plant pathogen systems (e.g., Chen et al, 2003 ; Bloemberg, 2007 ; Dunn et al, 2013 ; Hupp et al, 2019 ). Prior studies in our lab utilized fluorescent strains of Phytophthora capsici developed by Dunn et al (2013) to observe pathogen growth on cucumber fruit samples via fluorescence microscopy, and in a microtiter assay to test for effect of aqueous and methanolic extracts from cucumber fruit peel on pathogen growth ( Ando et al, 2015 ; Mansfeld et al, 2020 ). While microscopy allows for observations in vivo , quantitative assessment and multiple replications, if possible, are labor- and time-intensive.…”