Structure and Function

TWiM #306: Spirulina Smoothies

Podcast and Annotation Information
  • Annotation by Rachel Teich, Julia Borchers, DeAnna Wells, Kelsey Hudson, Remonda Fathalla, Emily Ngo, Mikaela Huerta, Ali Rustum, Jeremy Ritzert, Mel Melendrez-Vallard, Rebecca Seipelt-Thiemann, and Maggie Schlarman.
  • Podcast audio by TWiM: Listen to TWiM #306 Podcast
  • Podcast transcript by Otter. ai and edited by Harshita Sharma and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Klapper M, Hübner A, Ibrahim A, Wasmuth I, Borry M, Haensch VG, Zhang S, Al-Jammal WK, Suma H, Fellows Yates JA, Frangenberg J, Velsko IM, Chowdhury S, Herbst R, Bratovanov EV, Dahse HM, Horch T, Hertweck C, González Morales MR, Straus LG, Vilotijevic I, Warinner C, Stallforth P. 2023. Natural products from reconstructed bacterial genomes of the Middle and Upper Paleolithic. Science. 380(6645):619-624. doi: 10.1126/science.adf5300.
    • Wang S, He B, Wu H, Cai Q, Ramírez-Sánchez O, Abreu-Goodger C, Birch PRJ, Jin H. 2024. Plant mRNAs move into a fungal pathogen via extracellular vesicles to reduce infection. Cell Host Microbe. 32(1):93-105.e6. doi: 10.1016/j.chom.2023.11.020.

1. Paper Abstracts

1.1. Snippet paper discussion starts at 1:42 minutes

The Most Interesting Things (according to students)

  • The extraction of biosynthetic gene clusters from ancient dental calculus was interesting.
  • The authors were looking at teeth of Neanderthals and old human samples compared to current day humans rather than typical environmental samples and found photosynthetic organisms in the dental samples of the neanderthals; though there is skepticism as to whether this is due to consumption of contaminated water. The authors were able to produce an ancient product from the organism’s DNA that they found from the teeth. This unlocks the potential to produce products that may no longer be around and could potentially be beneficial to health.

The abstract cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

1.2. Main paper discussion starts at 22:56 minutes

The Most Interesting Things (according to students)

  • We could potentially deliver RNA into plant pathogens using vesicular transport to help control plant diseases.
  • mRNA transformation is very interesting because it is a cross between Type III and Type IV secretion. We had never heard of most of the techniques they used, so we thought it was interesting and clever how they were able to combine them into a coherent working experiment. The mRNA affects the mitochondria of the fungi inhibiting growth and reducing replication. This lets the plant defend itself against infection. This gives a new idea for potential plant disease solutions.

“Cross-kingdom small RNA trafficking between hosts and microbes modulates gene expression in the interacting partners during infection. However, whether other RNAs are also transferred is unclear. Here, we discover that host plant Arabidopsis thaliana delivers mRNAs via extracellular vesicles (EVs) into the fungal pathogen Botrytis cinerea. A fluorescent RNA aptamer reporter Broccoli system reveals host mRNAs in EVs and recipient fungal cells. Using translating ribosome affinity purification profiling and polysome analysis, we observe that delivered host mRNAs are translated in fungal cells. Ectopic expression of two transferred host mRNAs in B. cinerea shows that their proteins are detrimental to infection. Arabidopsis knockout mutants of the genes corresponding to these transferred mRNAs are more susceptible. Thus, plants have a strategy to reduce infection by transporting mRNAs into fungal cells. mRNAs transferred from plants to pathogenic fungi are translated to compromise infection, providing knowledge that helps combat crop diseases.” (Wang et al 2024, no changes)

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

Snippet Main
Vision and Change Topics
  • Metabolic Pathways (V&C_MP)
  • Evolution (V&C_E)
  • Information Flow and Genetics (V&C_IFG)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • Fundamental Statement 4 (ASM_4): Phylogenetic trees best reflect the evolutionary relatedness of all organisms, although microbial lineages may be difficult to define due to horizontal gene transfer or lack of conserved genes.
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • Fundamental Statement 16 (ASM_16): Genetic variation can influence microbial structures and their functions.
  • Fundamental Statement 7 (ASM_7): Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment.
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.
  • Fundamental Statement 25 (ASM_25): Microbes are used as models that provide fundamental knowledge about life processes.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Describe the value of metagenomic data to researchers and clinicians.
  • Identify the metric the researchers used to show successful metabolite production.
 S L
  • Propose an experiment based on a hypothesis generated in the podcast discussion.
 S H
  • Describe the molecular function and biological role of extracellular vesicles.
  • List the features that make Arabidopsis thaliana a good experimental organism.
  • Explain how extracellular vesicles (EV) protect Arabidopsis thaliana from fungal infection.
 M L
  • Predict the outcome of an experiment based on a hypothetical plant-fungal interaction based on this Arabidopsis thaliana fungal infection model.
 M H

1 Papers: Snippet (S) or Main (M)

2 Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

  • Metagenome Assembly (1:42–6:18): Metagenome reconstruction is the process of reconstructing genomes from metagenomic sequencing data. It’s a key tool in microbiome medicine, which aims to understand how microbial communities impact human health. Scientists are combining tools for metagenome assembly with computational tools to predict the existence and nature of biosynthetic clusters.
  • Metagenome Analysis (9:13–10:40): By looking at the number of lesions that were generated over time in the DNA of the Chlorobium, they were able to confirm that this was not a contaminant in the sample. The researchers were then interested in metabolite production of the bacteria and used a bioinformatics tool called antiSMASH (antibiotics & Secondary Metabolite Analysis Shell) to identify genes in the metagenome for producing metabolites. Through this process they found four gene clusters that make beneficial metabolites.
  • Metabolite Analysis (12:00–14:29): This analysis used nuclear magnetic resonance (NMR) and mass spectrometry (MS), which identify the chemical nature and abundance of molecules made by cultured microbes. The researchers inserted the ancient sequences by cloning three core genes into expression systems.  They used broad-host-range plasmids to determine which genes are needed in different biosynthetic clusters. They then use an engineered strain of Pseudomonas protegens (called an expression chassis) and another bacterium, Photorhabdus, to ensure the biosynthetic products are specific to ancient Chlorobia, not a combination of the two organisms. After a three-liter fermentation, they used NMR and MS to analyze the metabolite compounds’ structures, which are difficult to determine despite knowing their molecular formula.

4.2. Main Paper

  • RNA Imaging/Tracking (31:25–33:29): In this assay (also called Broccoli), the movement of mRNAs within and between cells via within extracellular vesicles can be studied.  Broccoli is a RNA mimic of green fluorescent protein.  In this study, they used this system to follow host-derived RNAs inside vesicles and inside fungal pathogen cells.  This study employed Nicotiana benthamiana and Arabidopsis thaliana, genetically manipulable plant species as the hosts and Botrytis cinerea as the fungal pathogen.
  • Ribosome Analyses (39:53–41:09): In these analyses, researchers isolate ribosomes and then used them in to study plant-fungal molecular interactions.  First, they sequenced the mRNAs bound to ribosomes to determine which specific mRNAs are actively translated. They called this method TRAP-seq in the paper.  Here, they used this analysis to find that EV-transferred plant mRNAs are associated with the ribosomes which suggests they are being translated. They identified 320 plant protein-coding mRNAs, including defense response genes, which are highly enriched on the fungal ribosomes across multiple replicates. This confirms that the ribosome association is not due to contamination.  In a second method, they used a gradient centrifugation method on lysates to show that the RNAs are associated with many ribosomes, not just single ribosomes.
  • Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) (41:33–42:01): RT-PCR is a laboratory technique that converts RNA to DNA then amplifies DNA segments using specific primers.  It’s a fast and inexpensive way to make billions of copies of DNA from RNA, even from very small samples. In the paper, they perform RT-PCR across experiments to look for SAG21 and APS1 RNAs.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

  • Chlorobium (6:20–15:20): Photosynthetic organisms found in teeth that use both light and rocks, it grows on sulfide, using hydrogen sulfide as its electron donor.
  • Monophyletic Clade (9:14–10:00): a group of organisms that share a common ancestor and all of its descendants.
  • Metabolomics (12:00–14:29): The systematic study of the entire collection of small molecules, or metabolites, in a biological system. It aims to identify and quantify these metabolites to understand their roles in cellular processes, physiology, and disease.

5.2. Main Paper

  • Secretion (25:26–26:05): Here they discuss the differences between Type III and Type IV secretion and how it relates to current bacterial pathways.
  • Extracellular Vesicles (EVs) (26:15–28:07): These are membrane bound vesicles that can transport important items from the cell to the outside environment. In this case, plants are able to transport harmful mRNA into fungi to inhibit growth.

6. Podcast Questions

  1. What is the primary purpose of constructing a metagenome?
    1. To sequence the genome of a single reference organism
    2. To analyze the genetic material of an entire microbial community
    3. To modify the genome of individual species in a laboratory setting
    4. To study the physical structure of microbial cells in a population
  2. What method did the researchers use to confirm that the ancient biosynthetic clusters could produce compounds?
    1. They compared the genetic sequences for these genes to modern bacteria.
    2. They analyzed the samples using gas chromatography and plasmon resonance.
    3. They cloned and successfully expressed the core genes in different bacterial hosts.
    4. They observed the natural production of compounds in other ancient samples.
  3. Why is reconstructing ancient bacterial genomes useful for modern medicine?
    1. It helps scientists remove harmful genes from modern bacteria using replacement by ancient genetic sequences.
    2. It helps scientists create synthetic bacteria and fungi that mimic ancient pathogens for vaccine development.
    3. It helps scientists eliminate modern pathogenic bacterial strains and prevent ancient diseases from re-emerging.
    4. It helps scientists understand how bacteria have evolved and identify new metabolites that may have other uses.
  4. Bacterial metabolites can often inhibit the growth of other bacteria, which is how researchers found metabolites with antibiotic properties.  If the researchers wanted to test their metabolites for antibiotic function, what would be a good experiment for them to do?
    1. A disk diffusion assay; grow bacteria in a lawn on an agar plate with paper disks impregnated with different metabolites and monitor the zone of clearing.
    2. An enzyme-linked immunosorbent assay; make dilutions of the metabolite and incubate with antibodies to common antibiotics, look for a loss of signal.
    3. A bacterial competition assay; grow bacteria in a co-culture, one expressing and one not expressing the metabolite, and monitor the ratio of survivors.
    4. A reporter activation assay; use a reporter gene fused to the promoter for common antibiotic biosynthesis genes, look for activation with metabolite exposure.
  5. What is the function of extracellular vesicles, and in which processes are they involved?
    1. They transfer molecules from the cell membrane to the endosomes which recycles cellular proteins.
    2. They generate energy for the cell through using an inverse ATP dependent proton pump in the membrane.
    3. They transport toxin-type molecules outside the cell so the cells survive toxin-antitoxin environments.
    4. They facilitate cell communication by transferring molecules like proteins and RNA between cells.
  6. According to the podcasters, what are the features of Arabidopsis thaliana that made it a good choice for these experiments? [pick all that apply]
    1. It has a short reproduction cycle and long life span.
    2. It is easy to grow in the laboratory.
    3. It is resistant to nearly all fungal infections.
    4. It is drought-tolerant, so requires little water.
    5. It has a fully sequenced genome.
    6. It is relatively easy to manipulate genetically.
  7. What was the main finding of the study regarding plant extracellular vesicles (EV)?
    1. EV transport plant small RNAs and proteins which interact with fungal proteins to enhance fungal growth.
    2. EV transport plant mRNAs into fungal cells where they are translated into proteins that reduce fungal growth.
    3. EV transport recycled fungal proteins to host plant cells to enhance the ability of plant to make specific nutrients.
    4. EV transport toxins from the fungus to the plant host to enable the plant to resist infection from other fungi.
  8. You are studying another plant-fungal interaction and suspect that extracellular vesicles are involved in this interaction, but may be in both directions.  So, you engineer the plant to produce EV with either mRNAs encoding green fluorescent protein (GFP or the green fluorescent protein (GFP).  You additionally engineer the fungus to produce EV with either mRNAs encoding a red fluorescent protein (mCherry) or the red fluorescent protein mCherry.  You inoculate each plant with each fungus and find the results noted below.  What is your conclusion?
  Fungal EV: none Fungal EV: mRNA (mCherry) Fungal EV: protein (mCherry)
Plant EV: none no fluorescence in either red fluorescence in plant no fluorescence in either
Plant EV: mRNA (GFP) no fluorescence in either red fluorescence in plant  no fluorescence in either
Plant EV: protein (GFP) green fluorescence in fungus red fluorescence in plant; green fluorescence in fungus green fluorescence in fungus
    1. Plants transport both and fungi transport mRNAs.
    2. Plants transport proteins and fungi transport both.
    3. Plants transport proteins and fungi transport mRNAs.
    4. Plants transport both and fungi transport neither.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 4ABD and 5).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Describe the experimental design of these plant-fungal interaction experiments, listing control groups and the dependent and independent variables tested.
  • Explain how the TRAP-isolated RNA from fungal ribosomes and fluorescent microscopy data demonstrate plant-fungal interactions.
  • Analyze RNA, immunoblot, and fluorescent microscopy data to make conclusions about RNA transfer and the translation of plant and fungal genes during fungal infections.
Experimental Background (Wang et al., Figure 4ABD)

Like animals and microbes, plants and microbes can have symbiotic interactions that range from mutualistic to parasitic.  Also like animals, plants have evolved mechanisms to reduce the destruction inflicted by parasitic microbes. Previous, recent studies had shown that pathogens and hosts deliver small RNAs to each other to regulate gene expression to regulate or enhance virulence.  In this study, Wang et al. (2024) investigate the plant-fungal interactions between the plant model species Arabidopsis thaliana and a pathogenic fungus Botrytis cinerea looking at delivery of plant molecules to the fungus via plant extracellular vesicles. In prior experiments, the researchers had identified mRNAs that were delivered via extracellular vesicles to the fungus: SAG21, PRXIIC, APS1, and HEL, so they next wanted to investigate whether these plant mRNAs were being translated into proteins by the fungus.  They conducted a series of experiments that show specific steps related to translation.  First, they needed to determine which, if any plant mRNAs were attached to a ribosome, so they engineered fungal strain whose large ribosome subunit was tagged (BcRPL23-YFP).  They infected Arabidopsis leaves with this engineered strain and then utilized the tagged ribosome protein to capture all RNAs that were ribosome-associated (TRAP) or simply all fungal RNAs (input).  The control for this experiment was a mixture of uninfected plant RNA mixed with RNA from culture fungus, which also underwent TRAP-capture or was left as input.  Reverse-transcription polymerase chain reaction (RT-PCR) was then used to detect specific mRNAs in the four samples: control plant-fungus input, control plant-fungus TRAP, infected plant input, infected plant TRAP (panel A).  The next in this series of experiments aimed to examine which RNAs were actively translated, and not just bound by a single ribosome.  To do this, plant leaves were infected with the fungus and the fungal cells re-isolated and used to make lysates.  These lysates were then subjected to gradient centrifugation to separate polysome (RNAs with multiple ribosomes), monosomes (RNAs with a single ribosome), and ribosome subunits.  RT-PCR was again used to detect specific mRNAs in the ten gradient fractions and the input lysate (panel B, left side).  As a control, the experiment was repeated with the addition of puromycin to the lysate prior to the gradient centrifugation.  Puromycin is a translation inhibitor known to disrupt polysomes (panel B, right side).  Finally, to evaluate whether plant proteins encoded by RNAs delivered by extracellular vesicles were fully translated in the fungal cells, they constructed four transgenic plant lines: 1) a fusion of SAG21 and yellow fluorescent protein (SAG21-YFP), 2) a fusion of a mutant, non-translatable SAG21 and yellow fluorescent protein (mSAG21-YFP), 3) a fusion of APS1 and yellow fluorescent protein (APS1-YFP), and 4) a fusion of a mutant, non-translatable APS1 and yellow fluorescent protein (mAPS1-YFP). They isolated extracellular vesicles from these plants and incubated them with fungal hyphae.  Fluorescent and light microscopy were used to visualize the fungal cell structures and fluorescence (panel D).

RT-PCR agarose gels and microscopy images
Figure 4. “Figure 4. Plant mRNAs are translated in fungal cells (A) Full-length plant transcripts were detected in TRAP-isolated fungal ribosome fraction after 36 h infection (Infection) by B. cinerea expressing ribosomal subunit BcRPL23-YFP, but were not detected in in vitro cultured B. cinerea transgenic BcRPL23-YFP hyphae mixed with Col-0 (Control) (upper panels). Immunoblot shows that TRAP specifically pulls down BcRPL23-YFP from infected Col-0 tissue using a-GFP antibody beads. OEP6, GRF10, and PRO5 were used as plant control genes, Bc-actin as a pathogen control gene. DNA size markers are in base pair (bp) (A, B, and F). Protein size markers are in kilodaltons (KD) (A and F). (B) RT-PCR shows that the transferred Arabidopsis mRNAs were associated with B. cinerea polysomes. The transferred plant mRNAs were shifted from the polysome fractions to the monosome fractions upon puromycin treatment. Ten fractions of equal volume were collected from top to bottom of 15% to 55% sucrose gradients. Treatment of puromycin or not is as indicated (+ or  ). (C) Western blot analysis shows SAG21-YFP and APS1-YFP proteins were not detectable in the extracellular fractions, including apoplastic wash fluids (AWFs), the P100 EV fraction (EVs), or the supernatant of the P100 fraction (S). As a positive control, annexin1(ANN1)-YFP-tagged protein was secreted into AWFs and present in EVs. The abundantly secreted pathogen-related protein1 (PR1), absent in EVs, was used as a secretion control. TET8 native protein was used as a marker for EV containing fractions. S, supernatant after 100,000 3 g centrifugation. (D) The fluorescence signals of YFP-tagged SAG21 and APS1 proteins were observed in fungal cells only after incubation with EVs isolated from transgenic plants expressing SAG21-YFP or APS1-YFP for 24 h but not at 0 h. There was no fluorescence signal for mSAG21-YFP or mAPS1-YFP after co-incubation. EVs were isolated from corresponding Arabidopsis transgenic lines expressing SAG21-YFP, APS1-YFP, mSAG21-YFP, or mAPS1-YFP. Scale bars, 10 mm….” (Wang et al., images and text cropped to show panels A-D).

7.1.2. Questions

  1.  In which lane(s) of panel A will you find the RT-PCR results that show whether a mRNA is associated with a fungal ribosome? [pick all that apply]
    1. control input; lane 1
    2. control TRAP, lane 2
    3. infection input, lane 3
    4. infection TRAP, lane 4
  2. Which of the listed plant mRNAs are associated with a fungal ribosome (panel A)?
    1. SAG21
    2. QEP6
    3. PRXIIC
    4. PRO5
  3. Which fractions of the gradient fractionations are those where the most dense materials will be found (panel B)?
    1. input
    2. 1–2
    3. 5–6
    4. 9–10
  4. Which mRNAs are associated with polysomes (panel B, left side), and what does this indicate?
    1. SAG21, APS1, Bc-Actin; many ribosomes are found on these mRNAs.
    2. SAG21, APS1; these are the most dense RNAs because they are longer.
    3. All the RNAs; the input lane shows that all RNAs are associated with ribosomes.
    4. none; the RNAs are only found associated with single ribosomes.
  5. What is the purpose of puromycin treatment in the polysome profiling experiment (panel B right side)?
    1. To degrade non-translatable mRNAs
    2. To inhibit ribosomes assembly
    3. To dissociate ribosomes from mRNAs
    4. To capture monosome-RNA complexes
  6. How does puromycin treatment affect the distribution of ribosomes on mRNAs (panel B right side)?
    1. It reduces the number of monosomes and increases the number of polysomes for all RNAs.
    2. It reduces the number of polysomes and increases the number of monosomes for all RNAs.
    3. It eliminates all polysomes thereby increasing the number of monosomes for all RNAs.
    4. It eliminates all monosomes thereby increasing the number of polysomes for all RNAs.
  7. Four transgenic plant lines were constructed and used to make extracellular vesicles.  Which of the plant lines are controls for the fluorescent experiment shown in panel D?  [pick all that apply]
    1. SAG21-YFP
    2. mSAG21-YFP
    3. APS1-YFP
    4. mAPS1-YFP
  8. What do(es) the transgenic plant control(s) (panel D) tell you?
    1. It/they will show background fluorescence levels due to the natural fluorescent properties of the fungus.
    2. It/they will show which fluorescent molecules were delivered as proteins using the extracellular vesicles.
    3. It/they will show whether the fluorescent molecules are degraded or protected by the fungal environment.
    4. It/they will show successful transfer of plant mRNA and protein cargo using the extracellular vesicles.
  9. What does the fluorescent protein signal in fungal cells indicate (panel D)?
    1. Plant mRNAs are translated into protein inside fungal cells.
    2. Fungal cells have proteins that are highly similar to plant proteins.
    3. Plant proteins are acquired by fungal cells using vesicles.
    4. Fungal cells fluoresce naturally when they infect plant cells.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of experimental design, controls, and goals for these experiments.
  • Identify key features of plant infection and microscopy images, as well as bar plots.
  • Analyze the data and make conclusions about which plant genes impact fungal virulence.
  • Evaluate the data and make conclusions about the localization of plant-derived proteins in fungal cells.
  • Hypothesize the susceptibility of different plant genotypes B. cinerea infection based on these results.
Experimental Background (Wang et al., Figure 5)

Like animals and microbes, plants and microbes can have symbiotic interactions that range from mutualistic to parasitic.  Also like animals, plants have evolved mechanisms to reduce the destruction inflicted by parasitic microbes. Previous, recent studies had shown that pathogens and hosts deliver small RNAs to the other to regulate gene expression to regulate or enhance virulence.  In this study, Wang et al. (2024) investigate the plant-fungal interactions between the plant model species Arabidopsis thaliana and a pathogenic fungus Botrytis cinerea looking at delivery of plant molecules to the fungus via plant extracellular vesicles. In prior experiments, the researchers had identified mRNAs that were delivered via extracellular vesicles to the fungus: SAG21, PRXIIC, APS1, and HEL, and that these RNAs are translated by fungal ribosomes.  So they next wanted to investigate the effect of these plant mRNAs/proteins on the fungus. They focused on infection, fungal growth, and protein localization in their studies.  To assay the effect on infection and growth, they first constructed fungal strains that expressed a specific wild-type or mutant plant RNA fused to the coding region for yellow fluorescent protein: 1) a fusion of SAG21 and yellow fluorescent protein (SAG21-YFP), 2) a fusion of a mutant, non-translatable SAG21 and yellow fluorescent protein (mSAG21-YFP), 3) a fusion of APS1 and yellow fluorescent protein (APS1-YFP), 4) a fusion of a mutant, non-translatable APS1 and yellow fluorescent protein (mAPS1-YFP), or a control of yellow fluorescent protein alone (YFP). They infected Arabidopsis leaves with each strain and examined lesion size (SAG21 results in panel A; APS1 results in panel B).  Following this, they next wanted to determine how the plant proteins were able to impact fungal growth, so they performed fluorescent microscopy on each fungal strain to localize the protein inside the fungal cells relative to mitochondria, which were stained with MITO-ID (panel C).  As a final experiment, to show any impact on fungi was directly due to plant-derived materials, they constructed plant lines that had SAG21 deleted (sag21), SAG21 deleted and a wild-type-YFP version added back (Psag21::SAG21-YFP/sag21), APS1 deleted (aps1), and APS1 deleted and a wild-type-YFP version added back (Paps1::APS1-YFP/aps1).  They infected control Arabidopsis leaves (Col-0) or the engineered Arabidopsis leaves with wild-type fungus and examined lesion size (SAG21 results in panel D; APS1 results in panel E).  Please note that in biology, p levels of 0.05 or less indicate statistical significance.

Photos of leaves and their mitochondria alongside bar charts.
Figure 5: “Plant mRNAs in fungal cells reduce infection. (A) B. cinerea transformants expressing Arabidopsis SAG21-YFP under a constitutive promotor oliC in an intergenic region show reduced virulence compared with transformants expressing the mutated transcript mSAG21-YFP or control YFP. (B) B. cinerea transformants expressing APS1-YFP display reduced infection capability compared with transformants expressing mAPS1-YFP or control YFP. For A and B, relative lesion sizes were measured at 60 h post-infection. The data are presented as mean ± SD, n = 10 leaves from at least three replicates. Ordinary one-way ANOVA using Dunnett’s multiple comparisons test was conducted to identify statistically significant differences. Small black circles represent individual values. (C) SAG21-YFP and APS1-YFP, but not the free YFP, are localized in mitochondria in B. cinerea transformants ectopically expressing these tagged proteins. Ordinary one-way ANOVA using Dunnett’s multiple comparisons test was conducted to identify statistically significant differences. Quantification of mitochondrial morphology change is displayed in violin plots, The size of mitochondrial (n = 100) in B. cinerea transformants was measured using Image J. Scale bars, 5 μm. (D) Enhanced susceptibility to B. cinerea was observed in T-DNA insertion knockout line sag21 (SALK_099663). Complemented transgenic sag21 line expressing SAG21-YFP driven by its native promotor shows no significant difference in susceptibility with Col-0. (E) The APS1 T-DNA insertion knockout line (aps1, SALK_046518) shows enhanced susceptibility to B. cinerea. Complemented transgenic aps1 line expressing APS1-YFP driven by its native promotor shows no significant difference in susceptibility with Col-0. For (D and E), relative lesion sizes were measured at 60 h post-infection. The data are presented as mean ± SD, n = 10 leaves from at least three replicates. Ordinary one-way ANOVA using Dunnett’s multiple comparisons test was conducted to identify statistically significant differences. Small black circles represent individual values.”(Wang et al 2024, no changes).

 

7.2.2. Questions

  1. What is the main experimental goal for this set of experiments?
    1. To demonstrate that B. cinerea can deliver RNAs to Arabidopsis
    2. To assess the impact of specific plant genes on fungal infection
    3. To identify new fungal virulence genes in Arabidopsis thaliana
    4. To illustrate that proteins are transported via extracellular vesicles
  2. Good experimental design includes controls.  What control conditions were included in the fungal infection experiment regarding SAG21 (panel A)? What was the purpose of using this/these as a control(s)? [Pick all that apply]
    1. Fungus engineered to express YFP; it was used to show the infection/lesion level expected in the absence of any plant gene expression.
    2. Fungus engineered to express two different versions of SAG21-YFP; it compares the results for two different strains to show they behave alike.
    3. Fungus engineered to express the mutant SAG21-YFP (mSAG21-YFP); it was used to show expected results without translated plant gene expression.
    4. Fungus engineered to express YFP;  It was used as a baseline for quantifying symbiotic growth of the fungal hyphae in the absence of plant vesicles.
  3. Lesion size was quantified to generate the data in panels A, B, D, and E.  The data are displayed by bar plots. Match the bar plot feature with its description. [1 = mean; 2 = median; 3 = outlier; 4 = standard deviation; 5 = individual measures; 6= standard error; 7 = measure of statistical significance (or not)]
    1. ________ bar height
    2. ________ p
    3. ________ Whiskers
    4. ________ Circles
  4. Which plant gene, when expressed in fungi, impacts fungal infection, and in what direction is the change (panels A and B)?
    1. SAG21 and APS1; both enhance fungal infection.
    2. APS1 but not SAG21 enhances fungal infection.
    3. SAG21 but not APS1 reduces fungal infection.
    4. SAG21 and APS1; both reduce fungal infection.
  5. What does the yellow color in the fluorescent microscopy results (panel C) indicate?
    1. where the two fluorescent colors overlap
    2. where the SAG21-YFP protein is localized
    3. where the APS1-YFP protein is localized
    4. where the fungal mitochondria are located
  6. Based on the fluorescent microscopy results (panel C), where do plant-derived SAG21and APS1 proteins likely localize in fungal cells?  What is your evidence?
    1. Cytoplasm; the fluorescence excludes the MITO-ID localization except in YFP alone.
    2. Cytoplasm; the fluorescence excludes the MITO-ID localization for all conditions.
    3. Mitochondria; the fluorescence overlaps the MITO-ID localization except in YFP alone.
    4. Mitochondria; the fluorescence overlaps the MITO-ID localization for all conditions.
  7. When researchers add a wild-type or functional gene back into a organism that is mutant for that gene, we call that a complementation.  Which plant lines in panels D and E are complemented lines? [pick all that apply]
    1. Col-0
    2. sag21
    3. Psag21::SAG21-YFP/sag21
    4. aps1
    5. Paps1::APS1-YFP/aps1
  8. Based on the results for the plant lines with deleted genes, would a plant that is genetically unable for form extracellular vesicles be more or less susceptible to fungal infection by B. cinerea? What is your reasoning?
    1. Less susceptible; no plant RNAs could be delivered to enhance fungal infection.
    2. More susceptible; no plant RNAs could be delivered to inhibit fungal infection.
    3. Less susceptible; fungal RNAs would not be taken up by the plant cells.
    4. More susceptible; fungal toxins would accumulate without vesicle trafficking.

8. Paper Information and Licensing

8.1. Snippet paper

  • Klapper M, Hübner A, Ibrahim A, Wasmuth I, Borry M, Haensch VG, Zhang S, Al-Jammal WK, Suma H, Fellows Yates JA, Frangenberg J, Velsko IM, Chowdhury S, Herbst R, Bratovanov EV, Dahse HM, Horch T, Hertweck C, González Morales MR, Straus LG, Vilotijevic I, Warinner C, Stallforth P. 2023. Natural products from reconstructed bacterial genomes of the Middle and Upper Paleolithic. Science. doi: 10.1126/science.adf5300.
  • This article is not licensed for Creative Commons use; thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page: https://www.science.org/doi/10.1126/science.adf5300

8.2. Main paper

License

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Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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