INTRODUCTION

Asian soybean rust (ASR) is a foliar disease of soybeans which is caused by the fungal species Phakopsora pachyrhizi (Ph. Pachyrhizi). This is one of the most damaging soybean diseases in the world resulting in billions of dollars in losses. Ideally, breeding ASR resistant cultivars would be the most cost effective method to overcome this disease, but ASR has proven capable of adapting to such cultivars and overcoming their resistance.1, 2 New methods for creating ASR resistant soybean cultivars are therefore required. For a plant to defend itself against microbes, each cell must have the ability to recognize microbes and potential pathogens. The initial plant immune response is started with the recognition of these microbes by receptor proteins on the cell membrane. These proteins recognize pathogen- associated molecular patterns (PAMP’s) that are characteristic to groups of microbes. Upon the recognition of PAMP’S the plant initiates PAMP triggered immunity (PTI). (Figure 1.) PTI is a quick, efficient and diverse response to an unknown pathogen, with the goal of preventing the pathogen from further penetrating the plant tissue. 3 In Nicotiana benthamiana (benthamiana), Pseudomonas syringae pv tomato DC3000 (DC3000) can suppress PTI and quickly cause a hypersensitive response (HR). HR is characterized by tissue collapse and cell death. Established PTI in the leaf can limit the HR induced by DC3000. As plants evolved PTI as a means to recognize potential pathogens and slow their ingression, so to did the pathogens evolve mechanisms to suppress this PTI. In order for Ph. pachyrhizi to successfully survive in their hosts, it suppresses a plant’s defense response through the action of effectors that are delivered into host cells through a specialized secretion system.4 Effectors have the ability to suppress the plant’s defense response and are essential in allowing the fungi to proliferate in the plant host.

Identification of Candidate Effectors in Phakopsora pachyrhizi for Suppression of PAMP Triggered Immunity Jason Aker, Mingsheng Qi and Steve Whitham.

Department of Plant Pathology and Microbiology, Iowa State University.

OUTLINE OF WORK A bacterial assay was developed to screen for the functionality of 82 potential candidate effectors in Ph. pachyrhizi. Individual candidate effectors were cloned into a bacterial strain, Pseudomonas fluorescens EtHAn (Effector to Host Analyzer). Each of these candidate effectors were then inoculated into benthamiana. A seven hour time period was allowed for the EtHAn inoculation to induce PTI in the plant cells, before DC3000, was inoculated in an overlapping ring on the EtHAn inoculation. After 24 hours and 48 hours, candidate effector proteins, from Ph. pachyrhizi, were screened for their ability to suppress the PTI induced by EtHAn, allowing for a HR in the overlapped area induced by DC3000.

RESULTS

Of the 86 candidate effector proteins tested in the first trial, 17 demonstrated the ability to suppress PTI allowing DC3000 to induce HR. 6 of the candidates demonstrated a high level of confidence in their ability to suppress PTI. The remaining 11 suppressed PTI on at least half of the patches tested. (Table 1.)

The 17 candidates were retested in an attempt to verify the outcomes in trial 1. Of the 17

effectors retested, 8 demonstrated a very strong suppression of PTI on the majority of leaf patches allowing for HR, 4 demonstrated a strong suppression on at least half of the patches tested, and 5 showed no suppression on the leaf patches. (Figure 2.)

DISCUSSION

In an attempt to screen for functionality of Ph. pachyrhizi candidate effectors, it was hypothesized, these effectors ought to suppress the PTI of benthamiana, allowing DC3000 to induce HR in the leaf. Of 86 candidate effectors tested, 17 demonstrated the ability to suppress PTI for DC3000 to induce HR in the first trial. Only those effectors that showed HR in half to more of the inoculated patches were chosen to be retested as they demonstrated a strong ability to function as a PTI suppressor. In trial one the data shows that five of these effectors were capable of suppressing the PTI in all of the test patches. The remaining effectors only showed this ability in half or better of the patches. This allows for the question of why did these effectors function some of the time but not all of the time? There are many factors that could explain this. One important factor is that these effectors are used by Ph. pachyrhizi to prevent Glycine max, soy bean, from initiating a PTI response. In this particular assay benthamiana was used to screen the effectors function. It is possible that these effectors are conserved by benthamiana and will have the full capability of working in this system, or that their function may be limited in this system. Another factor could be that each of these effectors may be weak or strong suppressors, they may function in conjunction with each other and not independently, and they may also function to suppress a specific level of PTI initiated by the plant cell. These 17 effectors were retested to confirm the findings of the first trial. In the second trial there was confirmation of the data in some of the candidates, by either continuing to show very strong (++) HR in most or all leaf patches, strong (+) HR in at least half of the leaf patches, or little to no (-) HR in the leaf patches. For example, candidate 49 in the first trial showed suppression of PTI and induced HR by DC3000 in all six tested patches and in the second trial, it showed suppression of PTI and induced HR in 8 out of 9 patches. However, some of the effectors did not confirm the same amount of suppression in the second trial as in the first. For example candidate 77, showed an HR to no HR ratio in the first trial of 4 : 2, but in the second trial the ratio was 0:7. Again, questions can be raised about the differences in results between the two trials. One possible explanation is in the variability of leaves on each plant. Plants in trial one were older than in trial two and the leaves could possibly have developed a stronger immune system that a weaker effector, or an effector specific to soy bean, could not suppress. Each individual leaf also has a level of vigor to environmental conditions and potential pathogens that would make the leaf more susceptible to the DC3000. It is also possible that in both trials the test patches should have been given longer than 48 hours to show a stronger level of HR caused by DC3000. A third trial of this assay will need to be carefully conducted, by limiting these variables, to confirm the function of these effectors. Further investigation into the potential functionality of all of these Ph. pachyrhizi candidate effectors is still needed. Simply because this assay showed that 17 of the candidates have varying ability to suppress PTI (strong, weak or no ability) in benthamiana, to confirm these proteins as effectors for ASR, they must be tested using other assays to validate their function. Further study would need to be carried out to test their ability to suppress PTI in soy bean. Lack of functionality for most of these candidate effectors, in this assay, does not preclude the fact that they may indeed be effector. Several other assays exist that allow identification of effector functionality by different methods.

ACKNOWLEDGEMENTS I would like to thank Professor Whitham and Mingsheng Qi for the opportunity to partake in this study, as well as the members of the Whitham lab for their help and for making this a fun, educational and enjoyable experience. I would also like to thank Adah Leshem and the Plant Genomics Outreach program, funded by the National Science Foundation, which made this experience possible.

HYPOTHESIS

Some Ph. pachyrhizi effectors ought to suppress the PTI of benthamiana, allowing DC3000 to induce HR in the leaf.

REFERENCES 1Miles, MR et al (2006) Plant Health Progr. Online: 29 pp. 2Bonde, MR et al (2006) Plant Disease 90: 708-16. 3Nicaise, V., Roux, M., Zipfel, C. (2009) Plant Physiology 150(4): 1638-1647 4Rafiqi M, et al (2010) The Plant Cell. 22(6): 2017-2032.

A.) EtHAn bacteria cultures, containing the empty vector (negative control) and containing each of the individual candidate effector proteins, were prepared for inoculation into benthamiana B.) Plants were inoculated (with a 1 mL syringe) on the underside of the leaf to allow for infiltration through the stomata. A mark was drawn around the area of inoculation. Each leaf contained a negative control (top left on leaf) for comparison to each of three candidate effector inoculations. In the initial trial, three plants with two leaves per plant were inoculated for a total of six test patches. C.) Seven hours later, DC3000 was inoculated allowing the DC3000 patch to overlap the EtHAn patch. A dashed line was used to mark the area of DC3000 inoculation. D.) 24 hrs. later the phenotype (HR or no HR) of the overlapped area was observed and recorded. E.) 48 hrs. later the phenotype was again observed and recorded. Candidate effectors that showed positive results for the suppression of PTI were chosen to be retested in a second trial following the same procedure, with the exception that as many as three leaves per plant were inoculated (if possible) resulting in up to nine test patches.

Table 1. Trial 1 phenotype of area with possible PTI suppression. Of the 86-candidate effector tested, shown are those that suppressed PTI and induced HR on equal to or greater than half of the sampled patches. A level of confidence of the test results was assigned based on the ratio of HR to No HR (A high level of confidence in the results due to the greater majority of HR: No HR was assigned a 1, ratios closer to equal numbers of HR : No HR were assigned a 2) 6-9 patches were tested per candidate effector; however, patches were discarded if the negative control showed HR on that particular leaf. E10E was included in this list as only one patch was evaluated and was positive for HR.

Candidate Effector Protein 48 Hour HR Phenotype 48 Hour No HR Phenotype Confidence of Trial 1

E86E 9 0 1

E35E 6 0 1

E49E 6 0 1

E82E 5 1 1

E14E 4.5 0.5 1

E15E 4.5 0.5 1

E7E 4 2 2

E77E 4 2 2

E23E 3.5 1.5 2

E36E 3.5 2.5 2

E6E 3 3 2

E9E 3 3 2

E16E 3 2 2

E17E 3 3 2

E81E 3 3 2

E3E 2.5 2.5 2

E10E 1 0 1

Figure 2. Images represent level of HR (cell death) after 48 hours on benthamiana leaves for each of the 17 candidate effector tested in trial 2. In trial 2, 6- 9 patches of each effector were inoculated. After 48 hours the patches were evaluated and the effectors were categorized by the number of patches in which the effector suppressed the PTI allowing for DC3000 to induce HR in the cells of the leaf. Effectors assigned a “++” are characterized as displaying a strong HR on the majority of patches. Those assigned a “+” are characterized as displaying HR on roughly half of the patches tested. Effectors that displayed no HR in the second trial were assigned a “-”.

A. B. C. D. E.

Figure 1. PAMP triggered immunity (PTI) in plant cells. Plant cells contain a variety of receptor proteins on the cell membrane (ex. FLS2) that recognize molecular patterns on attached microbes (ex. EtHAn). These proteins initiate the first line of defense, PTI, to protect the cell form the potential pathogen. PTI is a general term referring to a diverse immune system responses a plant has to a pathogen.

EpE E3E -

E6E ++ E7E + E9E + E10E -

E14E ++ E15E - E16E + E17E +

E23E ++ E35E + E36E - E49E ++

E77E - E81E ++ E82E ++ E86E ++

EtHAn strains

DC3000 ?