Humidity assay for studying plant-pathogen interactions in miniature controlled discrete humidity environments with good throughput

Abstract

This paper reports a highly economical and accessible approach to generate different discrete relative humidity conditions in spatially separated wells of a modified multi-well plate for humidity assay of plant-pathogen interactions with good throughput. We demonstrated that a discrete humidity gradient could be formed within a few minutes and maintained over a period of a few days inside the device. The device consisted of a freeway channel in the top layer, multiple compartmented wells in the bottom layer, a water source, and a drying agent source. The combinational effects of evaporation, diffusion, and convection were synergized to establish the stable discrete humidity gradient. The device was employed to study visible and molecular disease phenotypes of soybean in responses to infection by Phytophthora sojae, an oomycete pathogen, under a set of humidity conditions, with two near-isogenic soybean lines, Williams and Williams 82, that differ for a Phytophthora resistance gene (Rps1-k). Our result showed that at 63% relative humidity, the transcript level of the defense gene GmPR1 was at minimum in the susceptible soybean line Williams and at maximal level in the resistant line Williams 82 following P. sojae CC5C infection. In addition, we investigated the effects of environmental temperature, dimensional and geometrical parameters, and other configurational factors on the ability of the device to generate miniature humidity environments. This work represents an exploratory effort to economically and efficiently manipulate humidity environments in a space-limited device and shows a great potential to facilitate humidity assay of plant seed germination and development, pathogen growth, and plant-pathogen interactions. Since the proposed device can be easily made, modified, and operated, it is believed that this present humidity manipulation technology will benefit many laboratories in the area of seed science, plant pathology, and plant-microbe biology, where humidity is an important factor that influences plant disease infection, establishment, and development. Published by AIP Publishing. [http://dx

Overexpression of a plasma membrane protein generated broad‐spectrum immunity in soybean

“…Towards understanding the possible mechanisms of enhanced SDS resistance among the transgenic soybean lines with overexpressed GmDR1 , we investigated if any of the two major plant hormones, SA‐ and JA‐mediated defence pathways, are altered among the transgenic plants. Transcript levels of the two GmPR1 homeologues , GmPR1‐1 and GmPR1‐2 (Xu et al ., 2016; Xu et al ., 2018; Zeng et al ., 2017), marker genes for the defence pathway regulated by SA, were investigated. Surprisingly, GmPR1‐1 , but not GmPR1‐2 , is constitutively induced over 120‐folds more among the transgenic soybean lines as compared to that in the nontransgenic Williams 82 control (Figure 2e,f).…”

Differential tolerance of Zymoseptoria tritici to altered optimal moisture conditions during the early stages of wheat infection

“…Differences in inoculation conditions might also explain the differences in symptom expression between series. One possible improvement to the experimental method would be to screen larger numbers of isolates in a more standardized way (making it easier to separate environmental and physiological responses), for instance by controlling and testing moisture conditions in vitro using the experimental devices proposed by Li et al (2014; different RH conditions formed within humidity chambers obtained with different glycerol solution concentrations, even though saturated saline solutions might be preferable due to the more stable relative humidity they induce) or Xu et al (2016; discrete RH gradient formed in spatially separated wells of a multi-well plate). This would have the advantage of countering the aforementioned technical difficulties and the effect of the host resistance × environmental response interaction on the expression of pathogenicity in isolates, although, in our experiment, the two Z. tritici populations appeared similarly aggressive on the cultivar ‘Taichung 29’.…”

“…Bernard et al , 2013; Boixel et al , 2019a) than for moisture phenotyping (e.g. Li et al , 2014; Xu et al , 2016). Assessments of the differences in response to temperature in vitro (tests conducted without interaction with the plant) are biologically meaningful but have to be compared with in planta responses (tests conducted in interaction with the plant), as done for instance by Boixel et al (2019a).…”

Microfluidic chip for automated screening of carbon dioxide conditions for microalgal cell growth

“…Microfluidics-based biotechnologies have been extensively developed to enable system miniaturization, automation, and parallelization of biochemical processes, 19 as well as highthroughput culture, manipulation, and detection of cells and microorganisms. [20][21][22][23][24][25][26] Several microfluidic devices have been reported for on-chip cultivation, analysis, and transformation of microalgal cells under various growth conditions. [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] For example, toxicity screening using marine microalgal cultures in a microfluidic device was reported.…”