​​ Figure 11. [download fu​ll size image​ of the student version] [download fu​ll size image​ of the instructor version]​

​ Figure 3. Genetic basis for the hooded phenotype. There is a 305-bp insertion in the dominant Kap allele that is not found in the recessive kap allele. This diagram illustrates the 305-bp tandem duplication within the fourth intron of the dominant allele (Kap). Exons are depicted by green boxes and introns by solid black lines. ATG start and TAG stop codons are shown. PCR primers flanking this region can be used to amplify 1,247- and 1,552-bp fragments for the recessive (kap) and dominant (Kap) alleles, respectively. Adapted from Giménez et al. (2021).​

Learning Objectives

Upon Completion of this experiment, students will be able to:

• Describe 3 different plant disease epidemics and their impact on human society.
• Draw the interaction between primers and template DNA, and explain the effect of changing template sequence on primer binding.
• Compare and contrast conserved and variable regions of a gene in terms of sequence and functional similarity.
  

Introduction: Historic Impact of Plant diseases

Plants Get Sick Too!

The role of plant diseases in agroecosystems is woefully underappreciated. In fact, the simple understanding that plants are critical components of the world around us is often overlooked (Achurra 2022, Wandersee and Schussler 1999). In reality, plants fall victim to many of the same types of biotic diseases that humans do: fungal diseases, bacterial diseases, viral diseases, and nematode diseases. The impact of plant diseases though is not often clear to consumers; disease damaged crops rarely make it to grocery store shelves. Thus, the impact of plant disease of food availability and cost is largely hidden from the consumer. Recognizing the importance of plant health is the first step in understanding its role as paramount to global food security. Below are three brief vignettes about plant disease outbreaks that had widespread impact on human health and/or the economy.​

Irish Potato Famine—1845

In 1845, Phytophthora infestans made its way to Europe from the United States and Mexico. P. infestans is an oomycete (a fungal-like protist) that presents as moldy spots on potato leaves but eventually infects the actual potato tuber, causing it to rot and turn to mush. Potato fields in Belgium, France, and Ireland were devastated. Ireland was hit hardest due to its almost complete dependence on the crop. It is estimated that approximately one-million people died from starvation and related disease while another 1-2 million people emigrated to escape the famine. Even today, this pathogen is responsible for billions of dollars in damage to crops each year (Fry et al., 2015).​

Southern Corn Leaf Blight—1970

In 1970, an epidemic of Cochliobolus heterostrophus spread throughout the southern United States and the Corn Belt. The fungus causes dark lesions to appear on leaves and can cause the corn ear and cob to rot. Some areas of the Corn Belt saw 50% reductions in yield. Corn shortages increased the price of corn and even increased the price of alternate crops when demand for livestock feed shifted from corn to other crops. Financial losses due to this epidemic are estimated at one billion dollars (Ullstrup 1972, Wise et al., 1999).​

Modern Threats: Ug99 Stem Rust

Ug99 is a lineage of Puccinia graminis tritici, a fungus that causes wheat stem rust. Ug99 was discovered in 1999 in Uganda, and currently 7 races of the lineage have been identified. Ug99 showed novel virulence against the resistance gene Sr31 (Li et al., 2019). Because of this, it is estimated that 90% of wheat varieties are susceptible to Ug99. Ug99 is also virulent on barley. Ug99 has been active in Africa and the Middle East and its chances of spreading to Asia are likely. Wheat currently accounts for approximately 20% of humanity's food supply, and demand is expected to increase 60% by 2050. Thus, pathogens such as Ug99 stem rust pose a serious threat by reducing food availability worldwide (Singh et al., 2011).

Introduction: Powdery Mildew Resistance Genes in Barley

Powdery mildews are obligate biotrophic pathogens that can only grow on their host plants, and not on artificial medium. The lifecycle of cereal (e.g., barley and wheat) powdery mildews takes about 7 days and starts when spores germinate on the leaf surface and penetrate the host epidermal cells. Once it reaches the lumen (space between the cell wall and cytoplasm) a haustorium develops (this is a finger-like structure which allows the fungi to obtain nutrients from the host cells). After the haustorium develops additional hyphae form and spread across the leaf surface to invade neighboring cells. Powdery mildew can then either generate asexual spores or combine with a different mating type for sexual reproduction of diverse spores. Since both forms of reproduction generate new spores, this fungus can spread to other plants and start the infection cycle again.​

OWB Resistant Versus Susceptible Plants

Barley plants dusted with the spores from previously infected (susceptible) plants will begin to show signs of infection within 4-5 days. In the photos in Figure 11, plants were inoculated with spores of powdery mildew isolate 5874 (AVR_a6) and photographed seven days later. Note that some plants display green and healthy leaves, while other plants show leaves coated with the fungus. The resistant plants carry an allele of Mildew locus a, Mla6, which encodes a protein that recognizes the corresponding avirulence effector, AVR_A6, in the pathogen (Halterman et al., 2001).

​​​​​ Figure 12. Comparison of different regions of the Mla gene. A, Conserved region of the Mla gene. The DNA sequences of multiple Mla alleles are lined up. B, Divergent region of the Mla gene.​

Alleles of the Mla gene encode a protein known as a nucleotide-binding leucine-rich repeat receptor (or NLR; Brabham et al., 2023, Bettgenhaeuser et al., 2021, Halterman et al., 2001, Seeholzer et al., 2010, Wei et al., 2002). Two sets of primers were designed for Mla6, an allele known to confer resistance to powdery mildew, and segregating in the OWB-ISS. The first set was designed in a conserved region, meaning that when the sequences of several different Mla alleles were aligned, the base pairs were identical between each allele at almost all locations, as illustrated in Figure 12A. Conserved regions are more common at the 5' end of the Mla gene. The image in Figure 12A shows the aligned sequences of seven different Mla alleles. Because the sequence is conserved in this region, when PCR is completed using primers from this region, the same bands are present for each plant because the same sequence is present at all alleles at this sequence.

In contrast, the second set of primers was designed to target a divergent region of DNA as shown in Figure 12B. Divergent regions are more common towards the 3' end of the coding sequence. The image shows a region of DNA in which there are dozens of differences in base pairs between the seven aligned alleles. Because there are significant differences between the sequences, when PCR is completed with divergent primers, only Mla6 is amplified so only Mla6 shows a band on the gel.

PCR of Mla6 Resistance Gene

To investigate differences between resistance and susceptibility, we will use two different sets of Mla primers to amplify different regions of the Mla6 allele (Halterman et al., 2001). This PCR amplification, along with the electrophoresis of the PCR products allows us to visualize the differences in DNA between barley plants that are resistant and those that are susceptible to powdery mildew.​

Materials

• ​Thermal Cycler, Vortex
• 1.5-ml Centrifuge Tubes
• Cup, Ice
• Micropipettes
• Pipette Tips
• Mla PCR Primers
• Molecular-Grade Water
•  DNA Template(s)
• Markers
• PCR Tubes with Taq Beads Protocol: 0.2-ml PCR Tubes with Taq DNA Polymerase Beads

Instructions

1. ​Obtain your DNA in the 2.0 ml microcentrifuge tube. Begin to thaw it out.
2. Obtain a PCR tube with a Taq Polymerase bead at the bottom and label it like the other tubes with your OWB #, class period, date, and initials (you can just use your initials instead of your full name since the tube is small). You should pick 2 OWB plants that were resistant to powdery mildew, and 2 that were susceptible. You will amplify each sample using two different primer sets. You should have 5 tubes:

1. Negative Control (no template)
2. Conserved Primer (OWB -resistant)
3. Divergent Primer (OWB -resistant)
4. Conserved Primer (OWB -susceptible)
5. Divergent Primer (OWB -susceptible)
   
3. ​ ​​​While you are waiting, put crushed ice in your cup.
4. Make sure the bead is at the bottom of the tube. Your instructor will add 24 µl of the Mla primer mix to your PCR tubes.
5. Add 1 µl of your DNA template to your PCR tube.
6. Vortex the tube until the bead fully dissolves and the solution is clear.
7. Tap the PCR tube on the table to get all the solution down to the bottom of the tube.
8. Store tubes on ice until instructed to transfer your tubes to the thermal cycler.​

Gel Electrophoresis: Mla PCR Investigation

Using the same protocol from Experiment #1 (Using gel Electrophoresis to Analyze PCR products), prepare your PCR products (10 uL) with loading dye (3 uL) on a piece of wax paper, labeling each before adding to the wells in an agarose gel.

Gel Map

 When all samples are loaded, run the gel electrophoresis at 70 volts for 1 hour 15 minutes. These conditions are optimal for the resolution of the DNA fragments; however, they can be adjusted to complete the run within a class period (e.g., 80 volts for 50 minutes).​

Discussion Questions (while the gel is running)

1. You are working with two sets of primers. One that binds to a region of the Mla gene that is very similar between different alleles (Conserved primers), while the other set binds to Mla6 in a region that differs greatly among the other Mla alleles (Divergent primers).
   
   
1. ​​Why do you think some regions of a protein are the same for all the alleles, while other areas of the protein differ greatly?
   
   Answer: Different regions (motifs) of a protein carry out different functions; some are conserved for binding to other (conserved) proteins or nucleic acids, and some are divergent to bind to very specific regions of complementary proteins.
   
   
2. Which set of primers (conserved or divergent) do you think will let us differentiate between resistant and susceptible plants in this test? Explain.
   
   Answer: Divergent primers bind to specific sequences among Mla alleles, which recognize specific effectors in different isolates of the pathogen. If the isolate has the complementary effector for a particular Mla allele (e.g., AVR_a6 and Mla6), the plant will be resistant. If not (e.g., AVR_a8 and Mla6; or AVR_a6 and Mla8), the plant will be susceptible.
   
   

2. Why do we include a sample for the PCR machine that does not include template DNA? Would you expect this sample to produce a detectible PCR product? Why or why not?
   
   ​Answer: A no-template control is used to check that amplification is specific for the question you are asking and not due to random contamination.
   
   
3. What could a plant do to resist infection? (Brainstorm ideas)
   
   Answer: Hint: think about how humans and other animals resist infection; do plants have those same mechanisms, or do they have unique processes to resist infection?
   
   

​​Overview​​Experiment 1​​Experiment 2
​iTAG Instructor’s Planning Resources
​​AppendixGlossary​​​
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