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letters to nature

NATURE | VOL A430 | 04 NOVEMBER 2013 | www.mcmaster.ca

Arabidopsis Thaliana with

unknown (A) defense system

is found to have neither Basal

nor R-gene resistance against

Pseudomonas syringae

Jeevika Goyal*, Pakeezah Saadat

1209306

*McMaster University, Department of Biology, 1280 Main Street West,

Hamilton, ON Canada L8S 4K1

…………………………………………………… Plants have developed various signaling pathways to respond to distinctive biotic and abiotic stimuli; which compensates for their immobility to escape the changing environment. Plants have layers of resistance systems that use these signaling pathways to recognize and inhibit (or destroy) the pathogens. This experiment focuses upon two of the resistance pathways used by Arabidopsis Thaliana: Basal and R gene- mediated resistance against Pseudomonas syringae. Both Basal and R gene – mediated resistance initiate a different response in plants, making it susceptible to pathogen. In this experiment, we examined the bacterial growth in plant strains, NahG and BG2-GUS (controls), with known defense system to determine the resistance pathway in unknown plant strain. NahG has neither Basal nor R gene resistance while BG2 – GUS has both R gene – mediated and Basal resistance. Results indicate that unknown (A) plant has neither R gene – mediated nor basal resistance; as results was no cell death observed, which is a characteristic of R gene – mediated response. Also, when solution prepared from inoculated leaves was plated, increased bacterial growth pattern was observed in the unknown. Both the results bore a similar pattern to results observed in NahG (negative control). Therefore, the two results demonstrate that unknown (A) lacks both Basal and R gene – mediated form of resistance. This study further opens doors to future experiments examining the effects of accumulation of salicylic acid in other forms of plant resistance pathways such as age – related resistance exhibited by A.Thaliana against P.syringae.

Basal Defence system is non – plant specific which allows plants to release various antimicrobial proteins and strengthen its cell wall, when it senses MAMPS ( Microbe-/ Pathogen- Assiociated Molecular Patterns) such as as Flagellin. However, many pathogens have evolved to suppress Basal resistance by producing plant specific effectors; making plants a more susceptible host for growth and reproduction. On the other hands, plants have also developed another key resistance pathway: R gene – mediated resistance. This is a much faster and efficient way of pathogen activity, as in this all it takes is one plant receptor to recognize one pathogen receptor to initiate a response, such as Hypersensitive Response (HR). HR is an R gene – mediated cell death, in

which cell commits sucide to prevent spreading of infection among plants.This halters bacterial growth; thereby maintaining plant’s immunity against that pathogen. This type of HR response is thought to be Salicylic Acid (SA) dependent reponse; as SA is believed to acts a signal molecule that stimulates appropriate gene expression in the nucleus.

In this experiment both these defence mechansims are explored to determine the pathogen resistance pathway present in unknown A. It is done by comparing responses against pathogenicity among the plants strains with known defence mechanism and unknown A. NahG virulent and avirluent are SA- deficinent; therefore, lacks R gene mediated and basal resistance. BG2-GUS avirulent contains both R gene mediated and basal; while virulent only has basal resistance. NahG plant strain acted as negative control while the BG2- GUS plant strain was the positive control. In the first part of the experiment, all plants were inoculated with Pst strain and the observations were made and pictures of the leaves were taken using the microcrope over the course of next week. The symptoms were analyzed to draw conclusions. In the second part of the experiment the solution made from the incoluated leaves was plated to observe the amount of bacterial growth in the plants. We also examined the gene defence gene expression in BG2 – GUS, to ensure that that the defence gene was expressed. It was done by using the presence of reporter gene (Gus A); which, upon expression, displays a blue colour in plants.

Results showed that unknown plant has neither basal or R gene – mediated resistance. The symptoms observed from the first week fo the experiment indicate yellowing and rotting of leaves in both virulent and avirulent plant line of unknown; with virulent being more infected. No cell death was observed, a characteristic of Hypersenstive Response (HR). The symptoms observed were similar to symptoms observed in NahG (negative control) virulent and avirulent; however, in NahG virulent were slightly more rotten than avirulent. In BG2 – GUS avirulent leaves, yellow spots were observed, which is believed to be cell death in those areas; which is a symptom of R gene mediated response. It was also the least rotten; thereby was the most resistant. However, similar to NahG, rotting was observed in virulent plant line of BG2 – GUS; however, rotting was less intense in comparison (Fig 1).

Figure 1: Disease symptoms observed in leaves, 3 days after inoculations. Pictures were taken using microscope. Pannel of pictures show symptoms of intensity of disease due to Pst stain infection. A) Shows NahG plantline infected, with virulent being more infected compared to avirulent. Leaves looks paler in comparison to BG2 - GUS B) shows the infection in BG2 – GUS. Avirulent shows yellows spots, indicating cell death while virulent shows yellowing of leaves C) shows disease symptoms in unknown; with virulent and avirulent being rotten and brown in color. Unknown leaves are also paler in comparison to BG2 – GUS. Images indicate a similar rotting pattern between NahG and unknown. BG2 – GUS, seems most resistant, with least amount of rotting.

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letters to nature

NATURE | VOL A430 | 04 NOVEMBER 2013 | www.mcmaster.ca

We also tested for the amount of bacterial growth, to obtain a more quantitative data. The amount of growth of Pst in NahG was 9.56 x 103 cfu/ leaf disc in avirulent while 5.83 x 103 cfu/leaf dic in virulent. In BG2 – GUS the observed bacterial growth was 1.25 x 103 in avirulent and 3.13 x 103 in virluent. Finally, unknown plant line saw most growth with avirulent being 8.75 x 103 and virulent being 1.45 x 104. The results (Fig 2) showed most growth in unknown, followed by NahG, with BG2 – GUS showing the least amount of bacterial growth. Among the three strains avirulent plant line have lower growth in comparison for virulent. This pattern is applicable to unknown and BG2 – GUS; however the pattern seems to be reversed in NahG. Consistent with the results observed in part 1 of the experimetnt, the bacterial growth pattern observed in NahG was similar to unknown; with BG2 – GUS showing the least amount of bacterial growth.

Figure 2: Bacterial Growth of Pst strain in various plant lines of Arabidopsis Thaliana. Graph wascreated using the 10-2 dilution of all three plant lines. Data indicates that BG2 – GUS is the most resistant plant line, as it was observed to have least amount of bacterial growth. On the other hand, unknown A and NahG are most susceptible because of their increased bacterial growth pattern; indicating a lack of defense system against Pst bacterial strains. The error bars on the graph depict the standard deviation in plants;

Last part of the experiment was to ensure proper R

gene expression. It was done using a Receptor gene (GUS) which is near the R gene; therefore, if R gene is expressed then recepton gene also be expressed, which gives leaf blue colouring upon expression ( Figure 3). The results obtained showed no colouring in NahG (negative control); while most intense blue in GUS – stained seedlings. BG2 – GUS avirulent has blue stainind while BG2 – GUS virulent is observed to depict the faint blue colouring near the petiole region; however, the rest of the region seems to be blue – stained free.

Figure 3: Panel depicting GUS stained leaves. A) No blue stain is observe in the NahG, indicating lack of R gene. B) Blue staining in seedling (positive control),

indicating the expression of receptor gene C) GUS staining observed in BG2 – GUS avirulent, indicating the expression of R gene and receptor gene. D) Faint blue colouring observed in BG2 – GUS virulent, indicating lack of both R gene and receptor gene.

Both the results from part 1 and 2 of the experiment

indicate that unknown A has neither basal or R gene mediated defence pathway; as there was no cell death observed (Fig 1) and the bacterial growth in unknown is highest among the three plant lines. This pattern was consistent with results observed in the negative control of the experiment, NahG, that lacks both forms of resistance as well. The reason NahG is concluded to have neither type of resistance present because it showed rotting over cell death. Cell death is a type of R gene – mediated response, which was observed in BG2 – GUS and not in NahG; thereby, indicating that BG2 – GUS has R gene mediated resistance pathway while NahG lacks this pathway. Also, NahG lacks SA, which is required for both basal and R – gene mediated response; and since unknown exhibits the same pattern as NahG, it is believed to lack to SA as well. This will make unknown more susceptible to the pathogen, which is accounted by the high bacteria count observed ( Fig 2); thereby further indicating that unknown lacks both the defense mechanisms. Another observation made during the experiment that the cell death on BG2 – GUS was spotted by yellow spots on the leaf. This is because of lower cell density in parenchyma, which decreases the chlorophyll content in cell. This could be due to P.syringae causing the cell death faster in that region, which would lower the cell density there; thereby decreasing the chlorophyll content and causing yellow pigmentation. This may also explain the palelessness in NahG and unknown leaves in comparison to BG2 – GUS. Since, both of the plant lines are more suscpetible,they will have lower cell density in the parenchyma region, resulting in yellowness in the leaves.The least amount of bacterial growth and the fact that BG2 – GUS avirulent plant line leaves are greener in comparison, indicate that it has both forms of resistance, as it showed the least susceptibility to the pathogen. On the other hand, BG2 – GUS virulent also lacks R gene mediated resistance pathway, as no cell death was observed. However, it has basal defense, as the bacterial colony count in virulent BG2 – GUS is least among the virulent bacterial count in other plant lines. GUS staining results also coincides with other results; as in NahG and BG2 – GUS virulent no colouring wsa observed, indicating lack of expression of R gene. BG2 – GUS avirulent, has R gene – mediated pathway, which is indicated by blue colouring (Fig 3), as the receptor gene is closer to the gene D3000 avrRpt2 and when the resistance gene is expressed, so is GUS A. NahG and BG2 – GUS did not show any stain because they both lack R – gene; therefore, no expression of R gene means no expression of the receptor gene. Since, the previous results in unknown bore a closer resemblance to NahG, then BG2 – GUS, it indicates that unknown has neither type of resistance against Pst pathogen.

Despite the consistent results, the amount of observed bacterial growth in both the controls, NahG (negative) and BG2 – GUS (positive), is less compared to expected bacterial growth in the controls (Figure 4). The reason behind the variance could be that the bacteria completed their cycle faster that normal, and entered death phase. The amount of disease depends on three factors (disease triangle): plant genotype, bacterial genotype and

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letters to nature

NATURE | VOL A430 | 04 NOVEMBER 2013 | www.mcmaster.ca

environmental factors. Since, plant and bacterial genotype is analogous in both cases, leaving environment to be the only variable factor. A possible explanation could be that environment in the lab was favourable to bacterial growth. Thereofore, most bacteria completed their life cycle and died, before they were plated; thereby explaining the differences in numbers.

Figure 4: Comparisson between expected and observed bacterial growth of Pst strain in the two controls. Table indicates that expected values are higher compared to the values observed. However, a consistent pattern among all the values is that NahG (negative control) must show higher growth compare to BG2 – GUS (positive control) Our results from the experiment conclude that unknown A lack both forms of resistance, basal and R gene – mediated resistance pathway. Symptoms observed from part one of the experiment and bacterial count from part two of the experiment indicate that results are similar to that of NahG ( negative control). When both the controls were Gus stained, only BG2 – GUS avirulent plant line showed blue staining, indicating the expression of R gene. BG2 – GUS virulent showed no colouring, but bacterial colony count in this plant line was the least among other virulent plant lines and it showed yellowing on the third day, as opposed to rotting; indicating that bacterial cycle completion was slower in this then the other virulent plant lines. This shows that BG2 – GUS virulent may have Basal resistance. Lastly, NahG plant leaves observed rotting, greater bacterial colony count upon plating and no GUS stainng; inidcating the lack of neither form of resistance pathway against Pst strain. Since, the result pattern between unknown A and NahG, is similar, it is concluded that unknown also lacks both form of resistances (Refer to supplementary results attaced to find the tabulated summary of the collected data). In the experiment, it was observed that Salicylic Acid (SA) deficient plant lines, lack both, Basal and R – gene mediated resistance. This experiment could be further extended to study the impacts of Salicylic acid on other forms of resistance such as Age related resistance in Arabidopsis Thaliana. Mutants with different concentrations of Salicylic acid can be studied to examine the effects and find the optimal amount of Salicylic acid required to fight targeted pathogen. This may be useful for agricultural purposes, as crops that are infected by pathogen can be provided with something similar to Salicylic acid to enhance plant's immunity to the pathogen; and increase crop yield.

Methods Inoculation of Pst and Pst avrRpt2 strains in different plant lines Your materials and methods information goes here, with subheadings. This is 8pt. font, Garamond or Times New Roman, and is smaller than the remainder of your text.

Bacterial Quntitation

Your materials and methods information goes here, with subheadings. This is 8pt. font, Garamond or Times New Roman, and is smaller than the remainder of your text.

Gene expression

Ackowledgements A special thanks to Dr. Daniel and Dr. Cameron, for their assistance in decephering the results; and on theory/ conceptual aspect of the experiment. We would also like to acknowledge Britney Boroweic and Sinah Lee for their assistance during the experiment. We would also like to thank Usfa and Max for sharing their Gus staining pictures

http://www.ehow.com/about_5380594_bacteria-life-cycle.html

http://www.ncbi.nlm.nih.gov/pubmed/12848826

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC161673/

Genotype Pst DC3000 ( Virulent)

Pst DC3000avrRpt2 (avirulent)

Expected NahG ≥107 cfu/ ld ≥107 cfu/ ld BG2 - GUS 106 – 107 cfu/ld 104 – 105 cfu/ ld

Observed NahG 5.83 x 103 cfu/ld 9.56 x 103 cfu/ld BG2 - GUS 3.13 x 103 cfu/ld 1.25 x 103 cfu/ld