Journal club: resistance to wheat disease through recognition of a protein

Introduction: Wheat yellow rust disease
Every year large wheat crop losses are caused by wheat yellow rust disease, a disease on wheat caused by the fungus Puccinia striiformis f. sp. tritici (Pst). This fungus is not able to make all plant species sick: Nicotiana benthamiana, a relative of tobacco and a much studied species, is resistant to it. This plant is resistant because it can recognize the fungus and activate an immune response. Understanding how this plant recognizes the invading fungus and thus becomes resistant will increase our chance of ever breeding a resistant wheat variety that is also resistant to this fungus.

Results: Recognition of a protein is the key to a resistant plant
In their paper, Dagvadorj and colleagues show that N. benthiamiana recognizes a small protein secreted by the fungus. Recognition of this protein results in the expression of genes that lead to the activation of defense responses in the plant. The defense responses result in reduced disease symptoms after pathogen attack. Future studies identifying the receptor on the plant cells that recognizes PstSCR1 will therefore provide material to improve disease resistance against a range of pathogens.

For the enthusiasts: How did the researchers get these results?
This paper follows up on another paper published in 2009 that looked among all the genes of Puccinia striiformis f. sp. tritici for genes that are expressed when the fungus is infecting a plant. They found fifteen genes that encode secreted proteins and end their paper with ‘[t]hese genes are candidates for further studies to determine their functions in wheat-Pst interactions.’

Dagvadorj and colleagues performed just such a study. Their study focuses on one of the fifteen genes, one that codes for PstSCR1, as a possible explanation for the resistance of N. benthiamiana to Pst. They first check whether PstSCR1 mRNA is produced when the fungus infects wheat. This is the case from 72 h to 8 days post infection. Next, they checked the effect of PstSCR1 on plant immunity. To do this, they expressed PstSCR1 on N. benthiama leaves before putting pathogens on the leaves. The infection, as measured by the lesion diameter on the plant or the number of spores produced by the pathogen, is reduced in leaves with PstSCR1 expression. One possible explanation for the reduced infection is activation of the plant immune response. To test this, the researchers checked mRNA levels of genes involved in the plant immune response after infiltration of the leaves by purified PstSCR1. Both defense genes that were checked, NbACRE31 and NbCYP71D20, were indeed activated by PstSCR1. Thus, the detection of this protein results in the activation of the plant immune response, which in turn results in resistance against the pathogen secreting the protein.

Discussed paper
Dagvadorj B, Ozketen AC, Andac A, Duggan C, Bozkurt TO, Akkaya MS (2017) A Puccinia sriiformis f. sp. tritici secreted protein activates plant immunity at the cell surface. Scientific reports 7: 1141
DOI: 10.1038/s41598-017-01100-z

Paper that precedes this one:
Yin C, Chen X, Wang X, Han Q, Kang Z, Hulbert SH (2009) Generation and analysis os expression sequence tags from haustoria of the wheat strpe rust fungus Puccinia striiformis f. sp. Tritici. BMC Genomics  10: 626

Disclaimer: blog posts in the category ‘journal club’ are not intended to cover the whole paper discussed. Instead, I discuss the parts that I think are most interesting for a general public. I try my utmost to prevent any mistakes in these blogs, I apologize in advance for any mistakes that I make anyway.

Journal club: heart development

Main messages
The three major cell types of the heart develop and mature at different time points during heart development.The precise development depends on the localization of a cell within the heart. A mutation in the gene NKX2.5 can disturb the developmental process in mice. This probably explains the heart defect of both newborn people and mice that have this mutation in their DNA.

Background and aim
At birth, our hearts are fully functional pumps, pumping oxygen-poor blood to the lungs and oxygen-rich blood from the lungs to the rest of the body. The blood flows into the heart in the two atria, which can be visualized as chambers. Upon contraction of the walls of these chambers, the blood is pushed into the next chambers: the ventricles. From there, the blood is pumped out of the heart to the rest of the body and lungs. To be able to perform it’s pumping function, the heart contains muscle cells, allowing the heart to contract, endothelial cells, lining the blood vessels and heart chambers like a kind of inside skin, and fibroblasts, making the support framework of the heart, the so-called the ‘extracellular matrix’. This complex structure needs to develop during only nine months of pregnancy. In their paper, DeLaughter and colleagues studied the expression of genes in the heart of embryonic and newborn mice to learn more about how a heart and its different cell types develop. Ultimately, this will help us understand how defects in certain genes result in heart defects in newborn babies.

The researchers determined the expression of genes using RNA sequencing. They determined gene expression at different time points during development: at embryonic day 9.5, 11.5, 14.5 and 18.5, on the day of birth and at 3, 7 and 21 days after birth. Interestingly, they did not study gene expression in the whole heart at once, but in single cells. As explained here, different cell types perform different functions because different genes are expressed in each of them. By studying gene expression in separate cells, the researchers could determine the cell type and maturity of each of the analyzed cells.

Results & discussion
The ratio of the different cell types changes over time
The researchers discovered that the proportion of endothelial cells was stable over development. In contrast, the number of fibroblasts increased from none at all at embryonic day 9.5 to a little less than half of all cells at birth. This increase was at the expense of the cardiomyocytes. The decrease in the percentage of cardiomyocytes is possibly due to the decrease in cell division of these cells: more than half of the cardiomyocytes expressed genes associated with cell division during the start of embryogenesis, but none expressed them 21 days after birth.

Cells in the different heart chambers and different development time points can be distinguished based on different gene expression patterns
Interestingly, gene expression of the cardiomyocytes from the atria was clearly different from that of the cardiomyocytes from the ventricles. Not only that, even the cells from the left and the right ventricle could be distinguished based on gene expression! The difference between the ventricles became less clear over time, indicating that the chambers first establish their own characteristics, after which all of the cardiomyocytes mature.
The samples obtained at different time points were also clearly different from one another. Interestingly, comparing gene expression data from human hearts to this mice data, showed that human gene expression in the heart shortly after birth corresponded to the mice data obtained at 1-3 days after birth.

A mutation causing heart defects in humans delays maturation of the heart cells
Finally, the researchers looked at mice with a defect in one of their genes: gene NKX2.5. Both humans and mice with a defect in this gene, so with a so-called mutation, develop hearts in which the four chambers are not fully separated. Heart cells of mice with this mutation showed gene expression differences compared to mice with healthy hearts. These differences indicated that the mutant hearts had much fewer cardiomyocyte cells that had reached maturity at birth. Interestingly, while NKX2.5 is expressed mostly in the cardiomycytes, the maturation of endothelial cells was also affected.

DeLaughter and his colleagues have given us a better idea how heart cells develop and mature in an embryo. They also showed how this information can be used to understand the cause of heart abnormalities present at birth.

DeLaughter DM, Bick AG, Wakimoto H, McKean D, Gorham JM, Kathiriya IS, Hinson JT, Hornsy J, Gray J, Pu W, Bruneau BG, Seidman JG, Seidman CE (2016) Single-cell resokution of temporatl gene expression during heart development. Developmental cell  39: 480-490.

Disclaimer: blog posts in the category ‘journal club’ are not intended to cover the whole paper discussed. Instead, I discuss the parts that I think are most interesting for a general public. I try my utmost to prevent any mistakes in these blogs, I apologize in advance for any mistakes that I make anyway.

Journal club: roots perceive light transmitted through a plant

Light greatly influences plant growth and development. For instance, light is needed to generate energy via photosynthesis, and light cues allow plants to respond to neighbors that might overshadow them in the near future. Roots also respond to light, by changing their response to gravity and their amount of branching. However, these effects were mostly shown in experiment conducted in laboratory settings, where plants are grown on petri dishes and the roots are exposed to light. In contrast, in natural settings light often does not reach the roots, since light only penetrates a few millimeters into the soil.

Lee and colleagues were interested in the effect of light on roots in natural settings. First, they want to check whether exposing a plant to light changes gene expression in the root. They show that both direct root exposure to light and exposure of only the aboveground part of the plant, the so-called shoot, induce changes in gene expression in the root. Thus, in roots of plants that were exposed to light either at the root or at the shoot, different parts of the genetic code were ‘read’ compared with roots of plants grown in the dark. The light-induced changes were not the same in the root versus shoot exposed plants. There is a set of genes that always changes after exposure to light, though, independent of the exposure localization. Among these genes is the gene encoding the protein HY5, which is involved in the response of plants to light. The researchers show that a plant mutated in this gene, that is, a plant without a functional HY5 gene, that is grown in soil is impaired in its root’s natural response to gravity.

A clue about the method of activation of HY5 came from plants mutated in light receptors.  Light receptors are proteins that start signaling networks after absorption of light. A plant that is mutated for the gene encoding the light receptor phytochrome B (phyB), i.e. a plant with a non-functional phyB, activates HY5 much less when the shoot is exposed to light than a wild-type plant. In consequence, the mutants have less HY5 protein in their roots. The researchers then asked themselves whether root- or shoot-localized activatin of phyB results in HY5 activation. To test this, they grafted – joined parts of two plants together to form a ‘new’ plant – both a phyB mutant root with a wild-type shoot and the other way around. They then exposed the shoots of these plants to light and monitored induction of HY5 in the root. Interestingly enough, a mutation in the light receptor in the shoot did not affect HY5 expression in the root, but a mutation in the root prevented HY5 expression. This indicates that perception of light in the root is the cause of HY5 induction.

The researchers considered two possible mechanisms for the induction of phyB and HY5 expression. First, compounds produced in response to light in the shoot might travel to the root to induce a response there. Second, light itself might be transported to the root. The researchers first show that the compounds known to travel through plants to control light-mediated responses do not induce the activation of phyB in roots of dark-grown plants. Next, they checked their light transmission hypothesis. Light was shown on plant segments consisting of both root and shoot tissue with an optic fiber. This light could be detected at the root end of the segment. Thus, light can travel through the plant and could thus be responsible for the activation of phyB in the plant root. If this is true, the search for a signal that ‘informs’ the root of the light-status of the shoot can be stopped: light is transported through plants and can thus activate any necessary responses all by itself.

Lee, H. et al. (2016) Stem-piped light activates phytochrome B to trigger light responses in Arabidopsis thaliana roots. Science Signaling 9, ra106. DOI: 10.1126/scisignal.aaf6530

Disclaimer: blog posts in the category ‘journal club’ are not intended to cover the whole paper discussed. Instead, I discuss the parts that I think are most interesting for a general public. I try my utmost to prevent any mistakes in these blogs, I apologize in advance for any mistakes that I make anyway.

Journal club: fighting disease

Even though we are constantly surrounded by disease-inducing particles, most of us are healthy most of the time. We owe this to our immune system, which can combat most diseases that we are faced with. Fighting disease is based on our immune system’s amazing ability to distinguish ‘self’ from ‘non-self’. It is because of this ability that immune cells generally do not combat our own cells, but immediately start immune responses once non-self particles are recognized. The specific immune response that is started depends on the type of immune cell and the particle encountered. Examples of immune responses are the production of toxic compounds and the activation of other immune cells.

So how are the intruders perceived in the first place? Invading particles are recognized by the so-called B cells and T cells. These immune cells have receptors in their cell membrane, the membrane that separates the inside of the cell from the surroundings. On the outside of the cell, these receptors have structures that recognize (parts of) non-self particles. You can visualize this as a lock and key, with the receptor structure being the lock and (part of) a certain invading particle the key. When the particle fits on a receptor, the receptor is activated, leading to structural changes of the inner part of the receptor. These structural changes start a signaling process that ultimately results in an immune response.

While this system works against many disease-inducing agents, we all know that our immune system is not always successful. One example in which the immune system is not always successful in clearing away the disease-inducer is cancer. Because of this, humans have developed several therapies to treat this disease. However, these do not always work. In their recently published paper Roybal and his coworkers describe their work on harnessing properties of the T cell to develop a new treatment method.

The treatment method developed by Roybal and his colleagues is based on making synthetic T cell receptors. The part of these receptors on the outside of the cell can be designed to recognize any molecule. Upon activation, the receptor gets cut in half at the membrane, releasing the part inside the cell. This part can be designed to activate almost any gene – that is, a part of the genetic code coding for a particular protein. In theory, an engineered T cell with this receptor will result in a very specific, human-defined response upon recognition of the targeted disease-inducing particle.

The researchers tested the method by developing a receptor that recognizes a certain part of cancer cells. T cells with this receptor in their membrane were then cocultured with cancer cells. To easily test whether the synthetic receptor can drive activation of a specific gene, the inner part of the receptor was first engineered to drive activation of a gene encoding a fluorescent protein. Thus, if the receptor functioned as hoped, cells encountering cancer cells would become fluorescent. Excitingly, when exposed to the cancer particles, and only then, these T cells indeed became fluorescent.

Of course making a T cell fluorescent does not directly help curing cancer. Therefore, the researchers next wanted to see whether recognition of the cancerous cells could lead to effective immune responses. Again, the receptor performed as hoped: upon activation it could drive the secretion of proteins that shape the immune response, so-called cytokines, and the differentiation of immune cells into cells with anti-tumor fates. Moreover, cells could be engineered to induce the secretion of several therapeutics, resulting in cancer cell death. This is all the more exciting because some of these therapeutics are non-functional or toxic in the human body when delivered by injection. When delivered by the synthetic T cells, they are only delivered close to the tumor, preventing the side-effects.

While promising, the experiments described so far were all conducted on cell cultures. The immune responses of the engineered T cells might thus not be beneficial or even activated at all in organisms. Therefore, as a final experiment described in their paper, the researchers tested whether the engineered T cells also produce therapeutic agents when injected into the blood of a mouse with cancer. Not many T cells ended up in the tumor, but those that got there, and only those, did secrete the intended therapeutic agent. In a second experiment with a T cell receptor driving secretion of a different therapeutic agent, injection of the T cells resulted in clearance of the tumor.

While more research is needed before engineered T cells can be used in humans, the results published are very promising for two reasons. First of all, engineered T cells deliver their therapeutic agents locally at the site of disease. This prevents possible toxic effects of the agent when present throughout the body and ensures high doses at the right place. Second, at least in theory the receptors can be engineered to recognize any particle and lead to the activation of any gene. Since the engineered cells travel throughout the body, this means that injection with synthetic T cells has potential to treat many diseases.

Roybal, K.T. et al (2016) Engineering T Cells with Customized Therapeutic
Response Programs Using Synthetic Notch Receptors. Cell 167, 419-423.

Disclaimer: blog posts in the category ‘journal club’ are not intended to cover the whole paper discussed. Instead, I discuss the parts that I think are most interesting for a general public. I try my utmost to prevent any mistakes in these blogs, I apologize in advance for any mistakes that I make anyway.

Journal club: Surviving flooding

Like human beings, plants need to control water uptake to survive. Unlike humans, they cannot simply move to a dryer place when water levels rise, or go to a water source when they are in danger of dehydration. Instead, they need to cope while standing still. Plants take up water mostly via transporters in their roots. These transporters are called aquaporins (aqua = water, porin = protein) and only let water pass through. Aquaporins are located in the membranes surrounding the root cells and thus allow water to pass through the cell layers to the interior of the root. When the water reaches the central cylinder it starts its travel upwards to the leaves.

The transporters in the root can be opened, closed or removed from the cell membrane depending on the amount of water in the environment. When there is very little water available, the plant needs a lot of water transporters. In case of flooding the opposite, as few transporters as possible, is not always advantageous. When the aboveground parts of the plant are also under water, little water uptake is advantageous, so the plant will indeed want to restrict root water permeability. However, when the aboveground parts of the plant are not under water, water uptake is still required, since water is lost to evaporation. Thus, the aboveground environment determines the root water permeability that leads to greatest fitness. It is therefore reasonable to suppose that plants in locations with different kinds of recurrent floods have adapted by different responses to root flooding.

In their recent paper, Shahzad and co-authors describe their search for genetic factors that are involved in the regulation of root water permeability. In other words, they wanted to find parts of the genome, the heritable ‘code’ that each organism contains, that regulate this process. To find these parts, they performed a method called ‘QTL mapping’ in the model plant Arabidopsis thaliana (thale cress). First, the researchers took two populations of thale cress that differed in root water permeability. They then crossed these populations, resulting in offspring with pieces of the genome from each of the parents. The root water permeability of this offspring can be intermediate between the two parents or something more extreme, that is, close to that of either of the parents. Next, the genetic code of all the offspring with an extreme phenotype is checked. This offspring will have either parent’s DNA at random at most sites of the genome. However, the piece of the genome that is correlated with root water permeability will come from the parent whose root water permeability was inherited. In this way, Shahzad and co-authors found a region in the genome that explained 15% of the observed differences in root water permeability. This region contained the code of several genes. They then changed the code of each these genes one by one to check which gene contributed to root water permeability. This led to the discovery of a gene that negatively affects root hydraulics, which they named Hydraulic Conductivity of Root 1 (HCR1). The activity of this gene is influenced by potassium and oxygen levels, thus integrating diverse signals in the soil.

This brings us back to the strategy decision of plants with flooded roots. To study the effect of HCR1 on a plant’s response to flooding, the researchers studied a wild-type cultivar of thale cress and plants from the same cultivar with a mutated, that is, nonfunctional, HCR1 gene. When only the roots of these plants are flooded, the mutant grows more and has a higher water content in the aboveground parts of the plant than the wild-type plant. This confirms their hypothesis that a decrease in root water permeability is not always advantageous when roots are flooded. Apparently, in the wild-type plants the decrease in root water permeability by HCR1 results in less water uptake while the aboveground parts of the plant loose water by evaporation, resulting in a dehydrated plant. In contrast, in plants recovering from submergence the wild-type plants outperform the mutants. This shows that HCR1 increases plant growth and shoot water content in plants recovering from submergence.

Thus, the researchers discovered a gene involved in root water permeability regulation: HCR1. In further experiments they show that this gene differentially affects a plant’s fitness in different flooding conditions in one specific cultivar. The researchers subsequently discover that cultivars of thale cress from different locations across the world differ in their code of HCR1. These populations also differ in root water permeability, suggesting that changes in the code of HCR1 are a way in which plants have adapted to different flooding conditions. Future work will have to show whether the correlation between differences in HCR1 and root water permeability in the different cultivars is indeed causal. In addition, it remains to be elucidated how HCR1 decreases root water permeability in the first place.

Shahzad, Z. et al. (2016) A Potassium-Dependent Oxygen Sensing Pathway Regulates Plant Root Hydraulics. Cell 167, 87–98.e14. DOI:

Disclaimer: blog posts in the category ‘journal club’ are not intended to cover the whole paper discussed. Instead, I discuss the parts that I think are most interesting for a general public. I try my utmost to prevent any mistakes in these blogs, I apologize in advance for any mistakes that I make anyway.