Rapid Detection and Quantification of Plant Innate Immunity Response Using Raman Spectroscopy

We have developed a rapid Raman spectroscopy-based method for the detection and quantification of early innate immunity responses in Arabidopsis and Choy Sum plants. Arabidopsis plants challenged with flg22 and elf18 elicitors could be differentiated from mock-treated plants by their Raman spectral fingerprints. From the difference Raman spectrum and the value of p at each Raman shift, we derived the Elicitor Response Index (ERI) as a quantitative measure of the response whereby a higher ERI value indicates a more significant elicitor-induced immune response. Among various Raman spectral bands contributing toward the ERI value, the most significant changes were observed in those associated with carotenoids and proteins. To validate these results, we investigated several characterized Arabidopsis pattern-triggered immunity (PTI) mutants. Compared to wild type (WT), positive regulatory mutants had ERI values close to zero, whereas negative regulatory mutants at early time points had higher ERI values. Similar to elicitor treatments, we derived an analogous Infection Response Index (IRI) as a quantitative measure to detect the early PTI response in Arabidopsis and Choy Sum plants infected with bacterial pathogens. The Raman spectral bands contributing toward a high IRI value were largely identical to the ERI Raman spectral bands. Raman spectroscopy is a convenient tool for rapid screening for Arabidopsis PTI mutants and may be suitable for the noninvasive and early diagnosis of pathogen-infected crop plants.

Root-TRAPR: a modular plant growth device to visualize root development and monitor growth parameters, as applied to an elicitor response of Cannabis sativa

Background Plant growth devices, for example rhizoponics, rhizoboxes, and ecosystem fabrication (EcoFAB) have been developed to facilitate studies of plant root morphology and plant-microbe interactions in controlled laboratory settings. However, several of these designs are suitable only for studying small model plants such as Arabidopsis thaliana and Brachypodium distachyon, and therefore require modification to be extended to larger plant species like crop plants. In addition, specific tools and technical skills required for fabricating these devices may not be available to researchers. Hence, this study aimed to establish an alternative protocol to generate a larger, modular and reusable plant growth device based on differently available resources. Results Root-TRAPR (Root-Transparent, Reusable, Affordable three-dimensional Printed Rhizo-hydroponic) system was successfully developed. It consists of two main parts, an internal root growth chamber and external structural frame. The internal root growth chamber is comprised of a polydimethylsiloxane (PDMS) gasket, microscope slide and acrylic sheet while the external frame is printed from a three-dimensional (3D) printer and secured with nylon screws. To test the efficiency and applicability of the system, industrial hemp (Cannabis sativa) was grown with or without exposure to chitosan, a well-known plant elicitor used for stimulating plant defense. Plant root morphology was clearly detected in the system and plant tissues were easily collected and processed to examine plant biological responses. Upon chitosan treatment, chitinase and peroxidase activities increased in root tissues (1.7- and 2.3-fold, respectively) and exudates (7.2- and 21.6-fold, respectively). Phytohormones related to plant growth and defense response were higher in root tissues as compared to the shoots. Additionally, within two weeks of observation, hemp plants exhibited dwarf growth in Root-TRAPR system, easing plant handling and allowing increased replication under limited growing space. Conclusion The Root-TRAPR system facilitates exploration of root morphology and root exudate of C. sativa under controlled conditions and at a smaller scale. The device is easy to fabricate and applicable for investigating plant responses toward elicitor challenge. This fabrication protocol is modifiable to suit other plants and can be adapted to study plant physiology in other biological contexts, such as plant responses against biotic and abiotic stresses.

WHIRLY1 Regulates HSP21.5A Expression to Promote Thermotolerance in Tomato

Heat stress poses a major threat to plant productivity and crop yields. The induction of heat shock proteins (HSPs) by heat shock factors is a principal defense response of plants exposed to heat stress. In this study, we identified and analyzed the heat stress-induced Whirly1 (SlWHY1) gene in tomato (Solanum lycopersicum). We generated various SlWHY1-overexpressing (OE) and SlWHY1-RNA interference (RNAi) lines to investigate the role of WHIRLY1 in thermotolerance. Compared with the wild type (WT), the OE lines showed less wilting, as reflected by their increased membrane stability and soluble sugar content and reduced reactive oxygen species (ROS) accumulation under heat stress. By contrast, RNAi lines with inhibited SlWHY1 expression showed the opposite phenotype and corresponding physiological indices under heat stress. The heat-induced gene SlHSP21.5A, encoding an endoplasmic reticulum-localized HSP, was upregulated in the OE lines and downregulated in the RNAi lines compared with the WT. RNAi-mediated inhibition of SlHSP21.5A expression also resulted in reduced membrane stability and soluble sugar content and increased ROS accumulation under heat stress compared with the WT. SlWHY1 binds to the elicitor response element-like element in the promoter of SlHSP21.5A to activate its transcription. These findings suggest that SlWHY1 promotes thermotolerance in tomato by regulating SlHSP21.5A expression.

The Mycorrhiza Fungus Piriformospora indica Induces Fast Root-Surface pH Signaling and Primes Systemic Alkalinization of the Leaf Apoplast Upon Powdery Mildew Infection

We analyze here, by noninvasive electrophysiology, local and systemic plant responses in the interaction of barley (Hordeum vulgare L.) with the root-colonizing basidiomycete Piriformospora indica. In the short term (seconds, minutes), a constant flow of P. indica chlamydospores along primary roots altered surface pH characteristics; whereas the root-hair zone transiently alkalized—a typical elicitor response—the elongation zone acidified, indicative of enhanced H+ extrusion and plasma membrane H+ ATPase stimulation. Eight to 10 min after treating roots with chlamydospores, the apoplastic pH of leaves began to acidify, which contrasts with observations of an alkalinization response to various stressors and microbe-associated molecular patterns (MAMPs). In the long term (days), plants with P. indica-colonized roots responded to inoculation with the leaf-pathogenic powdery mildew fungus Blumeria graminis f. sp. hordei with a leaf apoplastic pH increase of about 2, while the leaf apoplast of noncolonized barley responded to B. graminis f. sp. hordei merely with a pH increase of 0.8. The strong apoplastic pH response is reminiscent of B. graminis f. sp. hordei–triggered pH shifts in resistance gene–mediated resistant barley leaves or upon treatment with a chemical resistance inducer. In contrast, the MAMP N-acetylchito-octaose did not induce resistance to B. graminis f. sp. hordei and did not trigger the primed apoplastic pH shift. We speculate that the primed pH increase is indicative of and supports the potentiated systemic response to B. graminis f. sp. hordei–induced by P. indica in barley.