Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - HOPESEE (How do pathogens exploit the symplast to promote disease?)

Teaser

It is well established that pathogens invade plant cells to establish an infection. Part of a biotrophic pathogen’s infection strategy is to secrete their own protein effectors into host cells to manipulate the host cellular processes to the pathogen’s benefit. It has been...

Summary

It is well established that pathogens invade plant cells to establish an infection. Part of a biotrophic pathogen’s infection strategy is to secrete their own protein effectors into host cells to manipulate the host cellular processes to the pathogen’s benefit. It has been observed that some effectors can move from cell-to-cell in the host and this suggests that a pathogen can invade and exploit cells surrounding the immediate infection site. To further support this hypothesis, my host lab observed that pathogen-induced plasmodesmata (PD) closure is suppressed in some virulent infections, suggesting pathogens attempt to keep intercellular connections open; some pathogens must maintain intercellular symplastic connectivity by counteracting pathogen-induced PD closure to promote disease. This proposes the question: why does a pathogen want PD open? We hypothesise that open PD allow pathogen effectors to move into non-infected cells where they target host processes to promote infection and allow pathogen access host resources such as sugars. In this study, I will use the Hyaloperonospora arabidopsidis (Hpa) – Arabidopsis interaction to identify cell-to-cell mobile effectors and their host targets. I will identify global changes of host gene regulation that are associated with cell-to-cell mobile effectors and examine the effect of cell-to-cell mobile effectors on sucrose transport around an infection site. The results will give a new insight into how pathogens exploit the symplast and noninfected cells to promote infection, characterising a poorly considered element of plant-pathogen interactions.
Overall objectives of HOPESEE are as follows:
1. How does Hpa access the host symplast? I will perform a large-scale screen of Hpa effectors to identify those that have intercellular mobility. To understand how cell-to-cell mobile effectors contribute to infection I will assess their impact on virulence and identify host targets for selected effectors.
2. How does Hpa manipulate non-infected cells? My host lab has recently developed a line in which infected cells are symplastically isolated from neighbouring, non-infected cells. I will compare global gene expression in this line and wild-type plants to identify host processes that are targeted by symplast mobile effectors. In a targeted approach I will further examine the regulation of genes associated with sucrose transport and distribution around infected cells and use novel cell biological tools to assess sugars (specifically sucrose) transport during infection.

Work performed

From the 475 predicted Hpa effectors (containing RxLR, RxLL and RxLCRN motifs), I selected 77 with high expression at early stage of the infection for synthesis. These were cloned as GFP fusion proteins via modular Golden Gate cloning in to plant expression vectors to examine their subcellular localization in N. benthamiana leaves. I developed a ‘2-in-1’ expression system to quantify the mobility of effector proteins in vivo and quantified the mobility of 19 effectors. I identified 9 effectors with greater than expected (‘super-mobile’) mobility effectors. I selected the 5 effectors with higher expression levels during infection for further analysis. This screen has established a comprehensive catalogue of Hpa effector localisations and cell-to-cell mobilities
Hpa is not transformable. Thus, to validate the role in virulence of our selected super-mobile effectors, we have generated transgenic Arabidopsis lines that overexpress these effectors with the aim to examine their impact on Hpa infection. To reduce any effect of the effector on development of the host, we have also generated inducible promoter (estradiol) lines for effector expression. To knockdown the effectors, we aim to use a HIGS system. We have generated host transgenic lines carrying dsRNA constructs which will allow us to examine if these lines are more resistant to Hpa. These tools are made and in the final stages of selection for homozygous lines suitable for virulence assays.
To investigate how host gene expression changes are affected by symplastic connectivity, we used RNAseq to compare transcriptional changes in lines in which we can manipulate PD aperture. Initially, we assayed two PD-closed lines: DMR6-cals3m and Est-PLUG. In contrast to our expectations, both lines were more susceptible to Hpa. Therefore, we changed our strategy and used the pdlp1,2,3 mutant which is more susceptible to Hpa and does not induce callose deposition in the walls of infected cells. We performed RNAseq on these mutants and were surprised to see that there were greater differences between mutants and Col-0 during development than during infection. However, we have identified and validated the differential regulation of the phosphate transporter PHT1;1 which raises new hypotheses about nutrient distribution when the symplast is mis-regulated.
To investigate infection-triggered activation of sucrose transporters, we generated a SUC2pro:GUS construct and assayed for expression in N.benthamiana leaves in the presence and absence of different cell-to-cell mobile effectors. Preliminary data showed that most effectors induced SUC2 expression and therefore there are likely effects of the transient expression system. I decided this objective would be best pursued in stable transgenic plants.

Final results

1. I categorised Hpa effectors according cell-to-cell mobility. My work has established a list of effectors that are candidates for regulating non-cell autonomous infection processes. Further, I have established tools and protocols for this type of analysis that can be exploited for similar investigations in any host-microbe interaction. The ‘2-in-1’ expression system for cell-to-cell mobility assays is an excellent tool to validate in vivo mobility and therefore of significant value and application to the research community.
2. Of the identified super-mobile effectors, there are 3 effectors that are larger than the size exclusion limit of PD of Benthamiana. This suggests some effectors move actively across the PD by manipulating PD aperture by themselves.
3. I have a generated list of Hpa RxLL and RxLCRN effectors that localise to different subcellular compartments. These have previously not been examined for subcellular localisation. I observed effectors localized to the endoplasmic reticulum, plasma membrane, nucleus, cytoplasm and the chloroplasts and thus I generated a list of candidate effectors that might interfere with organelle-specific processes. Of note is our identification of a chloroplast-localized effector that localises at starch granule initiation sites.
4. Our RNAseq data identified that PHT1;1 is mis-regulated during infection when PD are not closed. This suggests that phosphate transport and distribution is sensitive to the connectivity of the symplast and that it is a critical nutrient during infection. This opens a new avenue of investigation.

Website & more info

More info: http://n.a..