Functional Analysis of Barley Powdery Mildew Effector Candidates and Identification of their Barley Targets

Ali Abdurehim Ahmed

Functional Analysis of Barley Powdery Mildew Effector Candidates and Identification of their Barley Targets

Ali Abdurehim Ahmed

Abstract

The genome of barley powdery mildew fungus (Blumeria graminis f. sp. hordei, Bgh) encodes around 500 Candidate Secreted Effector Proteins (CSEPs), which are believed to be delivered to the barley cells either to interfere with plant defence and/or promote nutrient uptake. So far, little is known about the function of many CSEPs in virulence and the identities of their host targets. In this PhD study, we investigated the function of nine CSEPs and found that CSEP0081, CSEP0105, CSEP0162 and CSEP0254 act as effectors by promoting the Bgh infection success. Independent silencing of these CSEPs significantly decreased the Bgh haustorial formation rate, indicating their contribution to fungal pathogenesis. All these CSEPs were highly expressed during and after haustorial formation, suggesting that they play a role in virulence during the late infection processes. In addition, they all localized to the cytosol and the nucleus of barley epidermal cells. Furthermore, CSEP0162 and CSEP0254 accumulated in the extrahaustorial matrix in Bgh-infected cells. This implies that their virulence targets may localize in the same cellular compartments. Using yeast two-hybrid screens, two barley small heat shock proteins (sHsps), Hsp16.9 and Hsp17.5, were identified as interactors for both CSEP0105 and CSEP0162. These interactions were confirmed in planta by BiFC and co-localization studies. Small heat shock proteins are highly conserved ATP-independent chaperones that protect the cell from stress-induced protein misfolding and aggregation. Through their chaperone activity, some sHsps contribute to pathogen defence by stabilizing intracellular proteins, including resistance and defence signalling proteins. In this study, we validated the chaperone activity of the barley Hsp16.9, which prevented the aggregation of Escherichia coli cellular proteins during thermal stress. Interestingly, we could show for the first time that a microbial effector can inhibit the chaperone activity of a sHsp. Thus, CSEP0105 essentially stopped the activity of Hsp16.9. In summary, we propose a model where Bgh secretes these four effectors into the barley cell cytosol, nucleus and/or extrahaustorial matrix to interfere with the function of small heat shock protein machinery and other defence components to suppress plant immunity.

Originalsprog	Engelsk

Forlag	Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
Status	Udgivet - 2014

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PhD thesis - Ali Abdurehim AhmedAccepteret manuskript, 10,7 MB

https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01008973788&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK

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@phdthesis{366233713bca43228b34d90a34665790,

title = "Functional Analysis of Barley Powdery Mildew Effector Candidates and Identification of their Barley Targets",

abstract = "The genome of barley powdery mildew fungus (Blumeria graminis f. sp. hordei, Bgh) encodes around 500 Candidate Secreted Effector Proteins (CSEPs), which are believed to be delivered to the barley cells either to interfere with plant defence and/or promote nutrient uptake. So far, little is known about the function of many CSEPs in virulence and the identities of their host targets. In this PhD study, we investigated the function of nine CSEPs and found that CSEP0081, CSEP0105, CSEP0162 and CSEP0254 act as effectors by promoting the Bgh infection success. Independent silencing of these CSEPs significantly decreased the Bgh haustorial formation rate, indicating their contribution to fungal pathogenesis. All these CSEPs were highly expressed during and after haustorial formation, suggesting that they play a role in virulence during the late infection processes. In addition, they all localized to the cytosol and the nucleus of barley epidermal cells. Furthermore, CSEP0162 and CSEP0254 accumulated in the extrahaustorial matrix in Bgh-infected cells. This implies that their virulence targets may localize in the same cellular compartments. Using yeast two-hybrid screens, two barley small heat shock proteins (sHsps), Hsp16.9 and Hsp17.5, were identified as interactors for both CSEP0105 and CSEP0162. These interactions were confirmed in planta by BiFC and co-localization studies. Small heat shock proteins are highly conserved ATP-independent chaperones that protect the cell from stress-induced protein misfolding and aggregation. Through their chaperone activity, some sHsps contribute to pathogen defence by stabilizing intracellular proteins, including resistance and defence signalling proteins. In this study, we validated the chaperone activity of the barley Hsp16.9, which prevented the aggregation of Escherichia coli cellular proteins during thermal stress. Interestingly, we could show for the first time that a microbial effector can inhibit the chaperone activity of a sHsp. Thus, CSEP0105 essentially stopped the activity of Hsp16.9. In summary, we propose a model where Bgh secretes these four effectors into the barley cell cytosol, nucleus and/or extrahaustorial matrix to interfere with the function of small heat shock protein machinery and other defence components to suppress plant immunity.",

author = "Ahmed, {Ali Abdurehim}",

year = "2014",

language = "English",

publisher = "Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen",

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TY - BOOK

T1 - Functional Analysis of Barley Powdery Mildew Effector Candidates and Identification of their Barley Targets

AU - Ahmed, Ali Abdurehim

PY - 2014

Y1 - 2014

N2 - The genome of barley powdery mildew fungus (Blumeria graminis f. sp. hordei, Bgh) encodes around 500 Candidate Secreted Effector Proteins (CSEPs), which are believed to be delivered to the barley cells either to interfere with plant defence and/or promote nutrient uptake. So far, little is known about the function of many CSEPs in virulence and the identities of their host targets. In this PhD study, we investigated the function of nine CSEPs and found that CSEP0081, CSEP0105, CSEP0162 and CSEP0254 act as effectors by promoting the Bgh infection success. Independent silencing of these CSEPs significantly decreased the Bgh haustorial formation rate, indicating their contribution to fungal pathogenesis. All these CSEPs were highly expressed during and after haustorial formation, suggesting that they play a role in virulence during the late infection processes. In addition, they all localized to the cytosol and the nucleus of barley epidermal cells. Furthermore, CSEP0162 and CSEP0254 accumulated in the extrahaustorial matrix in Bgh-infected cells. This implies that their virulence targets may localize in the same cellular compartments. Using yeast two-hybrid screens, two barley small heat shock proteins (sHsps), Hsp16.9 and Hsp17.5, were identified as interactors for both CSEP0105 and CSEP0162. These interactions were confirmed in planta by BiFC and co-localization studies. Small heat shock proteins are highly conserved ATP-independent chaperones that protect the cell from stress-induced protein misfolding and aggregation. Through their chaperone activity, some sHsps contribute to pathogen defence by stabilizing intracellular proteins, including resistance and defence signalling proteins. In this study, we validated the chaperone activity of the barley Hsp16.9, which prevented the aggregation of Escherichia coli cellular proteins during thermal stress. Interestingly, we could show for the first time that a microbial effector can inhibit the chaperone activity of a sHsp. Thus, CSEP0105 essentially stopped the activity of Hsp16.9. In summary, we propose a model where Bgh secretes these four effectors into the barley cell cytosol, nucleus and/or extrahaustorial matrix to interfere with the function of small heat shock protein machinery and other defence components to suppress plant immunity.

AB - The genome of barley powdery mildew fungus (Blumeria graminis f. sp. hordei, Bgh) encodes around 500 Candidate Secreted Effector Proteins (CSEPs), which are believed to be delivered to the barley cells either to interfere with plant defence and/or promote nutrient uptake. So far, little is known about the function of many CSEPs in virulence and the identities of their host targets. In this PhD study, we investigated the function of nine CSEPs and found that CSEP0081, CSEP0105, CSEP0162 and CSEP0254 act as effectors by promoting the Bgh infection success. Independent silencing of these CSEPs significantly decreased the Bgh haustorial formation rate, indicating their contribution to fungal pathogenesis. All these CSEPs were highly expressed during and after haustorial formation, suggesting that they play a role in virulence during the late infection processes. In addition, they all localized to the cytosol and the nucleus of barley epidermal cells. Furthermore, CSEP0162 and CSEP0254 accumulated in the extrahaustorial matrix in Bgh-infected cells. This implies that their virulence targets may localize in the same cellular compartments. Using yeast two-hybrid screens, two barley small heat shock proteins (sHsps), Hsp16.9 and Hsp17.5, were identified as interactors for both CSEP0105 and CSEP0162. These interactions were confirmed in planta by BiFC and co-localization studies. Small heat shock proteins are highly conserved ATP-independent chaperones that protect the cell from stress-induced protein misfolding and aggregation. Through their chaperone activity, some sHsps contribute to pathogen defence by stabilizing intracellular proteins, including resistance and defence signalling proteins. In this study, we validated the chaperone activity of the barley Hsp16.9, which prevented the aggregation of Escherichia coli cellular proteins during thermal stress. Interestingly, we could show for the first time that a microbial effector can inhibit the chaperone activity of a sHsp. Thus, CSEP0105 essentially stopped the activity of Hsp16.9. In summary, we propose a model where Bgh secretes these four effectors into the barley cell cytosol, nucleus and/or extrahaustorial matrix to interfere with the function of small heat shock protein machinery and other defence components to suppress plant immunity.

UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01008973788&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK

M3 - Ph.D. thesis

BT - Functional Analysis of Barley Powdery Mildew Effector Candidates and Identification of their Barley Targets

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -