This study transformed the wheat blast pathogen Magnaporthe oryzae with fluorescent proteins to observe the infection process in wheat plants. Using fluorescence microscopy, the researchers were able to track fungal penetration, growth, and reproduction over time. They found that M. oryzae forms germ tubes and appressoria similar to related rice pathogens. Live imaging of infected millet leaves showed the fungus spreading from the spore tip into host tissue. Understanding the infection process could help identify genes involved in resistance.

1. An analysis of the Magnaporthe oryzae infection process in
wheat blast disease
Rachel D. Capouya, Dr. Thomas K. Mitchell
ABSTRACT
Wheat blast is a devastating disease of wheat caused by the
fungal pathogen Magnaporthe oryzae, Triticum pathotype.
Discovered in 1985, the spread of wheat blast in South
America in recent years poses a threat to the United States
wheat industry and has fostered a need for better
understanding of the pathogen’s interactions with its host.
While much is known about the biology of other
Magnaporthe species, namely those that cause rice blast
disease, there remains a dearth of information about the
intricacies of the wheat blast pathosystem. We have
transformed the closest known native relative of the wheat
blast pathogen, Magnaporthe oryzae Lolium pathotype, the
causative agent of grey left spot disease on turfgrass, with
two fluorescent proteins, GFP and tdTomato, driven by the
strong ToxA promoter. Using these fluorescing fungi, we
infected susceptible wheat plants and observed the process
of infection at multiple stages in the fungal and wheat life
cycles. By examining the pathogen’s method of penetration,
infection, and reproduction of a grass host, we are able to
elucidate the histology and etiology of M. oryzae and make
strides towards discovering the basis of resistance and
control. Presented will be a detailed view into the pathogen’s
infection process over time, including an analysis of the
physical infection structures of the pathogen and points of
interaction of the pathogen and host, and the resulting
outlook for future progress with this pathosystem.
A) GFP-transformed hyphae and B) spores of M. oryzae Lolium pathotype. C)
tdTomato-transformed hyphae and D) spores of M. oryzae Lolium pathotype
Figure 3: Live Cell ImagingMETHODS
Transformation
•Isolates of M. oryzae Lolium were transformed with one of
two fluorescence-inducing plasmids, one housing the Green
Fluorescence Protein gene and the other housing a tdTomato
gene.
•Transformation was conducted via a PEG-mediated protoplast
transformation followed by selection using Hygromycin B.
•Successful transformants were confirmed using the Nikon
epifluoresence microscope. GFP was detected by excitation at
400nm and tdTomato was detected using a 700nm filter.
•Transformed lines were harvested as spore stocks on filter
paper and stored at -20°C. Active lines were maintained on
oatmeal agar with agar plug transfers every 7-14 days.
InductionofAppressoriumFormation
•A 1.0x102 spores/mL suspension in sterile H2O was placed in
droplets on the hydrophobic side of GelBond in a moist
chamber.
•Observation after 24 hours
InfectionAssays
•Detached millet leaf inoculations were performed using a
1.0x105 spores/mL suspension in 0.25% gelatin.
•Leaves from Setaria italica spp. viridis were inoculated by
both drop inoculations and saturated filter paper and kept in a
moist chamber.
•Additional inoculations were performed on 0.7% agar media.
•Inoculated leaves were examined 24h and 48h post infection
via epifluoresence microscopy and live cell imaging with the
Olympus MPE FV1000 Multiphoton Laser Scanning
Microscope. A 40X immersion lens with 6X optical zoom was
used to obtain images of the fungal structures within the plant
tissue.
CONCLUSIONS AND FUTURE WORK
Conclusions
•The completed work on Magnaporthe oryzae, Lolium pathotype
indicated that this pathogen indeed functions on millet in a
similar manner to its well-studied relative, M. oryzae Oryzae,
does on rice.
•Germ tube and appressorium are formed within 24 hours of
spore contact.
•Live cell imaging of infected millet leaves shows a similar
process at 2 days following infection.
•Possibility of a spreading of fluorescent protein from the spore
tip into the host.
FutureWork
•Perform similar assays on susceptible wheat plants to analyze
pathogen behavior and spread throughout the plant. This will be
performed using detached wheat tissue inoculations as well as
traditional spray inoculations.
•Live cell microscopy to fully track pathogen development
immediately following inoculation.
•Assess inoculated wheat tissue at several short intervals in the
24 hours following inoculation to develop a fuller visual
understanding of the infection process.
•Expand further into the pathogen life cycle to observe
movement through or between host cells as well as reproductive
structure formation.
•Use knowledge of the structure formation and invasion of host
tissue to identify key genes to investigate in order to find a
potential source of resistance or control.
ACKNOWLEDGEMENTS
Thank you to Dr. Veena Devi Ganeshan and Dominique Tate for all of your
help, insight, and encouragement.
Thank you Dr. Sara Cole for facilitating the live cell imaging and greatly
assisting with the editing and production of the presented images and video.
Figure 2: Appressorium Formation on GelBond
Figure 1: Generation of Fluorescent Transformants
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College of Food, Agriculture, and Environmental Sciences/Department of Plant Pathology
Please scan the QR code to view a 3D video rendering of
Fig. 3C
A B
C D
A) GFP-transformed M. oryzae Lolium spore germinating and forming an appressorium 24h
post-infection on S. italcia spp. viridis. B) tdTomato-transformed spore germinating on S.
italica spp. viridis two DPI. C) Germinating spores and formed appressorium (white arrow)
two DPI. D) Magnaporthe germination and growth throughout millet leaf, two DPI. E)
tdTomato spore and appressorium (within white rectangle) on millet two DPI. F) 3D
rendering of structures in Fig. 3E.
A B
C D
A) Appressoria formation observed 24 hours after placement on hydrophobic surface.
B) Fluorescent image of a transformed spore forming an appressorium.
A B
E F