This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.
Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.
Magnaporthe oryzae, the rice blast fungus, infects an assortment of grain crops, including barley, wheat, and rice1. This pathogen causes devastating diseases and poses a worldwide threat to these valuable crops, causing complete crop loss if not controlled. Many labs around the world focus on rice blast disease because of its global threat and its attributes as an excellent model for plant-fungal interactions2. It has been fully sequenced, and the genetics of its infective cycle, particularly the early events, have been established3,4. The life cycle begins with a spore germinating on a leaf surface, forming the specialized penetration structure called the appressorium. The appressorium penetrates the leaf tissue, and infection continues with the development of lesions which start the process of sporulation and spread disease4. Preventing any of these early events would drastically inhibit this devastating disease. Consequently, most current research on blast disease has been focused on the early infection steps, from the germinated conidia forming an appressorium to the development of the invasive hyphae and the biotrophic interfacial complex (BIC)5.
The vast amount of research on blast disease has been conducted in rice, even though M. oryzae is a significant pathogen for a variety of crops, and newly evolved strains are emerging as a global threat to wheat6. While rice is one of the top three staple crops used to feed the population, along with wheat and corn, barley is the fourth cereal grain in terms of livestock feed and beer production7. As the craft beer industry grows, so does the economic value of barley. There are distinct advantages of using M. oryzae and barley as a pathosystem to study blast disease. First, there are strains of M. oryzae that infect only barley, as well as strains that can infect multiple grass species. For example, 4091-5-8 infects primarily only barley, while Guy11 and 70-15 can infect both barley and rice8. These strains are genetically similar, and the infection process is comparable9. Second, under standard laboratory and greenhouse conditions, barley is easier to grow, as it doesn't have the complicated requirements of rice (concise temperature control, high humidity, specific light spectra). There are also imaging challenges with rice due to the hydrophobicity of the leaf surface, which barley does not exhibit10.
This protocol presents a simple method for isolating and effectively utilizing barley leaf sheaths for microscopic analysis of multiple infection stages, using common laboratory supplies and a smartphone for data collection. This method for the barley leaf sheath assay is adaptable for labs across the world as it requires minimal supplies, and yet provides a clear picture of the microscopic interaction between the pathogen and the first few cells it infects. Whereas pathogenicity assays, such as a spray or droplet inoculation, can provide a macro view of the pathogen's ability to form lesions, this assay allows the researcher to visualize specific steps of early infection, from pre-penetration events to colonization of epidermal cells. Further, researchers can easily compare infection with the wild-type fungus to infection with a mutant reduced in virulence.
1. Preparation of experimental materials
2. Staining process
3. Mounting and imaging process
4. Image assessment and counting using ImageJ (FIJI)
A depiction of the initial workflow for this technique is displayed in Figure 1. The sheaths were harvested from 14-day-old susceptible "Lacey" barley plants (H. vulgare). The conidia were harvested from 10-day-old sporulating M. oryzae OMA plates, with a conidial suspension prepared using sterile ddH2O for a final concentration of 5 x 104 spores per mL. The inoculum suspension was directly applied to the leaf sheaths, which were secured to sterile Petri plates. The plates were kept in a warm, humid chamber for 48 h without light. Following the incubation period, the leaf sheaths were stained with trypan blue and prepared for imaging.
Infection sites were imaged using a smartphone and smartphone microscope adapter. A minimum of 10 images were recorded for each of the tested strains of M. oryzae. The experiment was repeated three times for a minimum of 30 images for each fungal strain. Figure 2A shows the representative results of a successful sheath assay, along with unstained and improperly dyed images for reference.
Depending on the hypothesis, these images can be quantified and analyzed in a plethora of ways. For this experiment, at 48 h post-inoculation, the total number of live spores (germinated spores) were counted, along with the number of appressoria and the number of successfully infected cells. A collection of 2,000 randomly mutagenized M. oryzae strains in a barley-infecting background were generated in the lab. Pathogenicity assays using spray and drop inoculations revealed many mutants with reduced lesion size compared to the wild type (a common phenotype for M. oryzae mutants)11. To tease apart these phenotypes, it was hypothesized that the reduced lesion size was caused by inhibition of one of the early infection steps (spore germination, appressorial formation, penetration peg formation, initial epidermal cell colonization), which is most easily tested via the leaf sheath assay. A promising candidate from the mutagenesis project was identified using a forward genetic named J99A12. This mutant did not show sensitivity to either nitrogen-starved or reactive oxygen conditions during the screen. During follow-up experiments, J99A produced significant numbers of appressoria on a hydrophobic surface, but displayed reduced lesion size on live barley. When tested using the sheath assay, J99A successfully developed appressoria and penetration pegs, that penetrated the leaf sheath but did not produce invasive hyphae once inside, thus suggesting the infection stopped at the penetration peg (Figure 2B). Successfully infected cells were identified by the presence of infective hyphae inside the tissue of the leaf sheath. Comparing the number of appressoria to the number of infected cells provided a percentage of successfully infecting appressoria. For wild-type 4091-5-8, 87% of appressoria successfully invaded and colonized the cell, while in the mutant J99A, only 36% of appressoria had hyphae inside the cell12.
Figure 1: Leaf sheaths harvested from 10-day-old barley and carefully removed with tools cleaned in ethanol. Conidia are collected, and the concentration is adjusted to 0.5-1.0 x 105 per milliliter with sterile water. The isolated sheaths are taped inside of a 60 cm Petri plate, and the conidial suspension is loaded into the sheath. The inoculated samples are kept at room temperature, in the dark, with beakers of hot water for humidity. The samples are stained in 45% acetic acid + 0.1% trypan blue for 1-2 h at 40 °C, then rinsed in 60% glycerol three times for 48 h post-inoculation. Stained samples are mounted and imaged. Please click here to view a larger version of this figure.
Figure 2: Representative results from the staining protocol. (A) Deviations from the staining protocol can result in suboptimal results. The heat and acetic acid serve to gently soften the leaf tissue. The post-staining rinses in 60% glycerol not only remove the excess stain, but help reduce the light scattering caused by the leaf and improve the image quality. Scale bars = 50 µm. (B) Representative images showing the robustness in this assay to see failed penetration and subsequent infection attempts of J99A (arrowheads), compared to successful attempts of 4091WT that resulted in the production of invasive hyphae to the leaf tissue (arrows). Scale bars = 50 µm. All images were taken 48 h post-infection. Please click here to view a larger version of this figure.
There are many commonly used assays available to test M. oryzae strains that provide a macroscopic-level visual of a compatible or incompatible infection response, such as spray or droplet inoculations, and the use of rating systems to quantifylesion sizes13,14. Another common assay for M. oryzae is to test the ability of the pathogen to form its specialized penetration structure, the apppressorium15. Described here is an easy method for observing changes in early infection processes quickly and efficiently at the cellular level, in the more facile barley plant. This method is unique, in that it uses general lab equipment and a smartphone for data capture. This method negates the need for camera-equipped and computer-controlled microscopes, making this protocol affordable for any lab. Using this method, we were able to identify at which step mutant J99A infection was halted, a question previous experiments have been unable to clarify.
The infection process of M. oryzae in rice, particularly colonization of the first few epidermal cells, has been well-imaged using fluorescent proteins, markers, dyes, confocal imaging, and advanced microscopy16. These types of imaging experiments are expensive, time-consuming, and require specific expertise. Many labs, using homologous recombination, are able to create genetic mutants of M. oryzae to analyze individual gene’s roles in the infection cycle, but may not have access to the advanced equipment and expertise required to explore the underlying cell biology. This protocol is intended to help bridge this gap, by using only a compound light microscope and a smartphone to capture digital images and generate z-stack-type videos of fixed leaf sheath tissues. This method enables imaging a few cell layers into the tissue, capturing the invasive fungal hyphae for up to 48 h. The leaf sheath of rice and barley have similar characteristics; they are only a few cell layers thick and have less chloroplasts, making them easier to image. As stated above, barley is less hydrophobic and easier to grow than rice, and many strains of M. oryzae can cause infection on rice and barley, making this experiment an easy swap for the more complicated rice assays.
A few limitations of this method include using the video function of the smartphone to collect z-field data, as the z-increment for the frame rate is unknown. Another limitation is that it requires fixed tissue (not live cells). However, because of the speed and ease of the protocol, this limitation could be overcome by examining various time points post-infection.
One of the most crucial steps of the protocol is proper staining. Improper rinsing causes excess dye in the slide preparation, causing dye saturation, and making the pathogen tissue indistinguishable from the leaf tissue. Meanwhile, under-staining prevents the pathogen tissue from contrasting against the leaf tissue.
The aforementioned method is adaptable to many scientific questions, and can be used to assess fungal mutants, assess various degrees of genetics resistance in barley plants, and test the efficiency of previously applied fungal control methods. It is also possible to extend this method to other plant-pathogen interactions, particularly other monocot leaf sheaths.
The authors have nothing to disclose.
The authors acknowledge funding from the USDA-NIFA award 2016-67013-24816.
Acetic acid | Sigma-Aldrich | A6283 | |
Cell phone | Pixel 4A | Any smartphone with a rear facing camera that can be mounted in an a holder will suffice. | |
Cell phone Microscope adapter | Vankey | B01788LT3S | https://www.amazon.com/Vankey-Cellphone-Telescope-Binocular-Microscope/dp/B01788LT3S/ref=sr_1_2_sspa?keywords=vankey+cellphone+telescope+adapter+mount&qid=1662568182&sprefix= vankey+%2Caps%2C63&sr=8-2 -spons&psc=1&spLa=ZW5jcnlwd GVkUXVhbGlmaWVyPUFKNklBR jlCREJaMEcmZW5jcnlwdGVkSWQ 9QTA2MDMxNjhBRFYxQTMzNk9E M0YmZW5jcnlwdGVkQWRJZD1BM DQxMzAzOTMxNzI1TzE3M1ZGTEI md2lkZ2V0TmFtZT1zcF9hdGYmY WN0aW9uPWNsaWNrUmVkaXJlY3 QmZG9Ob3RMb2dDbGljaz10cnVl |
Glycerol | Sigma-Aldrich | G5516 | |
Microscope | AmScope | FM690TC | 40x–2500x Trinocular upright epi-fluorescence microscope |
Oatmeal old fashioned rolled oats | Quaker | N/A | https://www.amazon.com/Quaker-Oats-Old-Fashioned-Pack/dp/B00IIVBNK4/ref=asc_df_B00IIVBNK4/?tag=hyprod-20&linkCode=df0 &hvadid=312253390021&hvpos= &hvnetw=g&hvrand=98212627704 6839544&hvpone=&hvptwo=&hvq mt=&hvdev=c&hvdvcmdl=&hvlocint =&hvlocphy=9007494&hvtargid =pla-568492637928&psc=1 |
ProMix BX | ProMix | 1038500RG | |
Rectangular coverglass | Corning | CLS2975245 | |
Slides, microscope | Sigma-Aldrich | S8902 | |
Stage micrometer | OMAX | A36CALM7 | 0.1 mm and 0.01 mm Microscope calibration slide |
Trypan blue | Sigma-Aldrich | T6146 |