Plant and Cell Physiology Advance Access published June 4, 2014
Ectopic Expression of RESISTANCE TO POWDERY MILDEW8.1 Confers Resistance to
Fungal and Oomycete Pathogens in Arabidopsis
Running head: Expressing RPW8.1 defends two different pathogens
*Corresponding author: Dr. W.-M. Wang, Rice Research Institute, Sichuan Agricultural
University, Chengdu 611130, China; Tel, 86-28-86290949; Fax, 86-28-86290897; e-mail, Downloaded from http://pcp.oxfordjournals.org/ at Universite Laval on June 30, 2014
[email protected] Subject areas: environmental and stress responses
Colour figures: 8
Number of supplementary material: 2
© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail:
[email protected] 1
Ectopic Expression of RESISTANCE TO POWDERY MILDEW8.1 Confers Resistance to
Fungal and Oomycete Pathogens in Arabidopsis
Running head: Expressing RPW8.1 defends two different pathogens Xian-Feng Ma 1
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, Yan Li , Jin-Long Sun , Ting-Ting Wang , Fan Jing , Yang Lei , Yan-Yan 1
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Huang , Yong-Ju Xu , Ji-Qun Zhao , Shunyuan Xiao and Wen-Ming Wang * Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, China
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Institute of Bioscience and Biotechnology Research and Department of Plant Science and
Landscape Architecture, University of Maryland, College Park, MD 20850, USA 3
These authors contributed equally to the paper.
Abbreviations: Avr, avirulent factor; CC, coiled-coil; dpi, day post inoculation; EHC, encasement of haustorial complex; EHM, extrahaustorial membrane; eLRR, extra-cellular leucine-rich repeat; ETI, effector-triggered immunity; Gc, Golovinomyces cichoracearum; Hpa, Hyaloperonospora arabidopsidis; HR, hypersensitive response; LRR, leucine-rich repeat; LSCM, Laser scanning confocal microscopy; NBS, nucleotide-binding site; NP, native promoter; PAMP, pathogenassociated molecular pattern; PTI, PAMP-triggered immunity; R, resistance; RLK, receptor-like kinase; RLP, receptor-like transmembrane protein; RPW8, RESISTANCE TO POWDERY MILDEW 8; T, transgenic; TM, transmembrane; YFP, Yellow fluorescent protein; WT, wild type.
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Abstract Broad-spectrum disease resistance is a highly valuable trait in plant breeding and attracts special attention in research. The Arabidopsis gene locus RESISTANCE TO POWDERY MILDEW 8 (RPW8) contains two adjacent homologous genes, RPW8.1 and RPW8.2, and confers broadspectrum resistance to powdery mildew. Remarkably, the RPW8.2 protein is specifically localized to the extrahaustorial membrane (EHM) encasing the feeding structure of powdery mildew whereby RPW8.2 activates haustorium-targeted defenses. Here, we show that ectopic
leads to unique cell death lesions and enhances resistance to virulent fungal and oomycete pathogens that cause powdery mildew and downy mildew diseases, respectively. In powdery mildew infected plants, RPW8.1-YFP accumulates at higher levels in the mesophyll cells underneath the infected epidermal cells where RPW8.2-YFP is mainly expressed. This cell-typepreferential protein accumulation pattern largely correlates with that of H2O2 accumulation, suggesting that RPW8.1 may spatially collaborate with RPW8.2 in activation of resistance to powdery mildew. Interestingly, when ectopically expressed from the RPW8.2 promoter, RPW8.1YFP is also targeted to the EHM of powdery mildew and the transgenic plants display resistance to both powdery mildew and downy mildew. Using YFP as a reporter, we further reveal that the RPW8.1 promoter is constitutively active but induced to higher levels in cells at the infection site, whereas the RPW8.2 promoter is activated specifically in cells at the infection site. Taken together, our results suggest that RPW8.1 (and its promoter) is functionally distinct from RPW8.2 and may have a higher potential in engineering broad-spectrum resistance in plants.
Key words: Downy mildew — Epidermal cell — Extrahaustorial membrane — Haustorium — Mesophyll cell — Powdery mildew.
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expression of the yellow fluorescent protein (YFP)-tagged RPW8.1 from the native promoter
Introduction Plants use multiple layers of defense mechanisms to protect themselves from pathogenic microbes. In addition to the preformed barriers such as leaf hairs, rigid cell walls, pre-existing antimicrobial compounds, plants mainly exploit a two-layered induced immune system to counteract the invasion of potential pathogenic microbes and adapted pathogens (Jones and Dangl 2006). The first layer is activated when plant cell-surface receptors recognize pathogenassociated molecular patterns (PAMPs) of an invading microbe and initiate immune responses to
termed PAMP-triggered immunity (PTI), is suppressed by effectors of adapted pathogens that are delivered into host cells through the type III secretion system of many phytobacterial pathogens or secreted by haustoria or inter-/intra-cellular hyphae of filamentous pathogens (Dou and Zhou 2012). For the second layer of induced plant immunity, host intracellular receptors, historically called resistance (R) proteins, are able to specifically recognize their cognate effectors, e.g. avirulent factors (Avrs), and activate race-specific resistance that is also known as effectortriggered immunity (ETI) (Spoel and Dong 2012). ETI is often stronger than PTI and features with hypersensitive response (HR), a rapid localized cell death at the infection site. Numerous R genes have been cloned and functionally characterized in the past decades. Based on the deduced proteins, R proteins can be classified into three types (Jones and Dangl 2006; Xiao et al. 2008). First, most characterized R proteins are members of the nucleotidebinding site and leucine-rich repeat (NBS-LRR) superfamily acting as the intracellular immune receptors that directly or indirectly recognize specific pathogen effectors (Bonardi et al. 2012). Second, some R proteins possess an extra-cellular LRR (eLRR) domain such as cell-surfacelocalized receptor-like transmembrane proteins (RLPs) and receptor-like kinases (RLKs) (Dangl and Jones 2001; Jones et al. 1994). Third, some other R genes encode atypical R proteins that
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prevent infection (Gomez-Gomez and Boller 2000; Zipfel et al. 2006). This layer of defense,
are distinct from that of NBS-LRR or eLRR type R proteins (Jones and Dangl 2006; Xiao et al. 2008). For example, the wheat R protein PM21 is a Serine/Threonine kinase mediating broadspectrum and durable resistance to powdery mildew disease in wheat (Cao et al. 2011). Lr34, another wheat R protein, is a putative ATP-binding cassette transporter and confers resistance to both powdery mildew and rust fungal pathogens (Krattinger et al. 2009). The rice R protein Xa27 contains two putative transmembrane domains and is induced specifically at the infection site initiating resistance to Xanthomonas oryzae pv. oryzae (Xoo) strains harboring AvrXa27 (Gu et al.
The Arabidopsis R genes RPW8.1 and RPW8.2 (referred to as RPW8 in later text unless otherwise indicated) are two tandemly-arrayed homologous genes at the RPW8 locus from accession Ms-0 plants that show broad-spectrum resistance against all infectious powdery mildew isolates tested. A previous report showed that 35S promoter-based overexpression of RPW8.1 or RPW8.2 in accession Col-0 plants, which lack both genes and are susceptible to Gc, results in enhanced resistance against a broad range of powdery mildews including Gc (Xiao et al., 2001), indicating that each of the RPW8 genes can independently confer resistance to Gc. The RPW8 proteins are atypical R proteins in terms of protein structure: they are predicted to encode small proteins (18–20 kDa) with a putative N-terminal transmembrane (TM) domain and one or two coiled-coils (CCs) Xiao et al. 2001). Interestingly, the expression of RPW8.2 is induced by powdery mildew infection (Xiao et al. 2005) and the RPW8.2 protein is specifically targeted to the extrahaustorial membrane (EHM) that encases the fungal feeding structure, the haustorium. There, RPW8.2 activates defense responses such as callose deposition and H2O2 production through a yet unknown mechanism (Wang et al. 2009). Thus, adequate expression and precise EHM-localization of RPW8.2 are key to activating broad-spectrum resistance to powdery mildew (Wang et al. 2010; Wang et al. 2009). RPW8.1 shares 45% identity and 65%
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2009; Gu et al. 2005).
similarity with RPW8.2 in amino acid sequences and is found to localize around chloroplasts in mesophyll cells (Wang et al. 2007). Because powdery mildew pathogens only invade epidermal cells, the mesophyll cell-limited accumulation of RPW8.1 raises the questions as to how RPW8.1 contributes to resistance against powdery mildew in the epidermal cells and whether RPW8.1 could activate defense against pathogens such as Hyaloperonospora arabidopsidis (Hpa), which are capable of infecting mesophyll cells. In the present report, we show that ectopic expression of RPW8.1-YFP from its native promoter
oomycete (Hpa Noco2) pathogens. RPW8.1-YFP expressed from its native promoter accumulates in clusters of mesophyll cells. Upon powdery mildew infection, RPW8.1-YFP is further induced to a higher level in mesophyll cells beneath the infected epidermal cell, which contrasts with RPW8.2’s EHM-specific subcellular localization. Interestingly, when expressed by the RPW8.2 promoter, RPW8.1-YFP is also specifically targeted to the EHM in the infected epidermal cells, while retaining its accumulation in the mesophyll cells. Taken together, our results demonstrate that RPW8.1 is also capable of EHM-targeting but is functionally distinct from RPW8.2 in activating resistance against two different pathogens.
Results Ectopic expression of RPW8.1-YFP results in cell death distinct from that caused by RPW8.2-YFP The RPW8 resistance locus was isolated from Arabidopsis accession Ms-0. The Arabidopsis line Col-gl does not have RPW8.1 or RPW8.2, and thus is RPW8-null. Any phenotypes observed in transgenic Col-gl expressing RPW8 can be considered as the result of the expression of the transgene. Under this scenario, we generated transgenic (T) plants expressing RPW8.1-YFP
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or the RPW8.2 promoter confers enhanced resistance to virulent fungal (Gc UCSC1) and
from its native promoter (NP) and examined the phenotypes of six representative lines (R1Y1, R1Y2, R1Y3, R1Y4, R1Y5, and R1Y6) at T6 generation along with control transgenic line C15 expressing YFP from the constitutive promoter 35S (35S::YFP, wild type, WT). Compared to transgenic plants expressing 35S::YFP, all NP::RPW8.1-YFP transgenic lines examined were smaller in plant stature (Fig. 1A), of which R1Y1 was the smallest, R1Y6 was the biggest and R1Y4 was in the middle. Small pits were developed on the adaxial side (Fig. 1B) and little bulges at the same location were seen on the abaxial side of mature leaves from all NP::RPW8.1-YFP
death lesions caused by ectopic expression of NP::RPW8.2-YFP in the transgenic line R2Y4 (Fig. 1D). Trypan blue staining of pitted leaves of NP::RPW8.1-YFP transgenic plants (R1Y4) revealed the collapse of 3-5 epidermal cells and 10-20 mesophyll cells in the center of the pit, which was surrounded by long and narrow irregular epidermal cells in a radial array (Fig. 1E, 1F). Under higher magnification, we observed that irregularly shaped epidermal cells centered clusters of dead cells and the underlying mesophyll cells became smaller than those underneath healthy epidermal cells (Fig. 1F). In contrast, no cell-shape change was observed for cells surrounding the dead epidermal and mesophyll cells in leaves of NP::RPW8.2-YFP transgenic plants (R2Y4), though the dead mesophyll cells became smaller than the healthy ones (Fig. 1G). Additionally, H2O2 was accumulated at higher levels in the intercellular area next to the clustered dead or dying mesophyll cells in the NP::RPW8.1-YFP transgenic plants (R1Y4) (Fig. 1H). However, in NP::RPW8.2-YFP transgenic plants (R2Y4), H2O2 accumulated inside the mesophyll cell, but no changes in cell shape occurred around the dead cell (Fig. 1I). To test if the expression levels of RPW8.1-YFP correlate with the growth and disease reaction phenotypes of the six transgenic lines, we examined levels of the RPW8.1-YFP mRNA by quantitative real time PCR (qRT-PCR) and levels of the fusion protein by western blot analysis.
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transgenic lines (Fig. 1C). These confined small pits were visually different from HR-like cell
As shown in Supplementary Fig. S1, the lines R1Y1 and R1Y5 showed the highest levels in mRNA, while R1Y4 and R1Y6 showed the weakest. Similarly, R1Y1 showed the highest and R1Y6 the weakest, while the rest four lines showed roughly similar-levels of accumulation of the 44 kDa RPW8.1-YFP protein (Supplementary Fig. S1B). Thus, the high-level expression of RPW8.1-YFP appeared to be associated with reduced plant stature. Intriguingly, we detected a strong band of the YFP size (i.e. 27 kDa) in the western blot (Supplementary Fig. S1B arrow), implying that RPW8.1 might be cleaved from RPW8.1-YFP. However, the biological significance
To understand why ectopic expression of RPW8.1-YFP triggers cell death pits, we carefully examined the subcellular localization of RPW8.1-YFP expressed from the native promoter. While RPW8.1-YFP accumulated around chloroplasts in discrete clusters of mesophyll cells, just as previously reported (Wang et al. 2007), we further observed that RPW8.1-YFP signals often coincided with pit formation (Fig. 2A; Supplementary Fig. S2A). The coincidence of pit formation with the RPW8.1-YFP signal was more evident when the distorted shape of the neighboring cells was revealed by propidium iodide (PI) staining (Fig. 2B). More interestingly, we found that those chloroplasts surrounded by RPW8.1-YFP tended to cluster together, and some appeared to fuse with each other (Supplementary Fig. S2B). Such chloroplast aggregates partially or completely lost chlorophyll auto-fluorescence before cell demise (Supplementary Fig. S2C), implying that the merged individual chloroplasts were being disintegrated. These observations imply that discrete accumulation of RPW8.1-YFP in the periphery of chloroplasts may trigger the collapse of chloroplasts and subsequent cell death, which may further impact the shape of the surrounding cells, forming a pit in the leaf surface.
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of this cleavage is currently unknown and worthy of investigation in the future.
Ectopic expression of RPW8.1-YFP confers resistance against powdery mildew and downy mildew Chloroplast-associated localization of RPW8.1-YFP in mesophyll cells prompted us to test if ectopic expression of RPW8.1-YFP could activate resistance against pathogens invading mesophyll cells. To this end, we inoculated two-week-old seedlings of NP::RPW8.1-YFP transgenic lines with Hpa Noco2 which is virulent to transgenic Col-gl line C15 expressing 35S::YFP. Disease phenotypes were recorded at 6 days post inoculation (dpi). All six
Noco2 (Fig. 3A). We also examined these lines for powdery mildew resistance by inoculating Gc UCSC1 on six-week old plants and scored resistance phenotypes at 8 dpi. As shown in Fig. 3B, three (R1Y1, R1Y4 and R1Y5) of the six lines tested showed obvious enhanced resistance to powdery mildew disease. By contrast, transgenic plants expressing NP::RPW8.2-YFP (R2Y4) were fully susceptible to Hpa Noco2, although they were resistant to Gc UCSC1 (Fig. 3C-3F) as previously observed (Wang et al. 2009). Statistic analysis on data from spore counting indicated that,in addition to R1Y1, R1Y4 and RY5, R1Y2 and R1Y3 also exhibited enhanced resistance to powdery mildew (Fig. 3F), whereas R1Y6 was as susceptible to Gc UCSC1 as WT (Fig. 3B and 3F). Because R1Y4 exhibited the best resistant phenotypes to both powdery mildew and downy mildew, we focused on this line for further investigation. To understand how RPW8.1 activates resistance against two different types of pathogens, we firstly examined cell death in R1Y4 after inoculation of Hpa Noco2. Leaves of R1Y4 or WT infected by Hpa Noco2 were subjected to trypan blue staining at 6 dpi. Microscopic examination showed that, in most cases, spores landed in the long narrowed cells centering a group of collapsed cells where a pit was formed failed to germinate (Fig. 4A). Spores landed outside the pit area might germinate; however, the germinated spores either failed to penetrate the host cell
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independent lines (from R1Y1 to R1Y6) tested clearly showed enhanced resistance to Hpa
or triggered host cell death after penetration (Fig. 4B and 4C). In rare cases where the oomycete pathogen was able to develop intercellular hyphae and form haustoria in adjacent mesophyll cells, all haustorial complexes were besieged by a callosic encasement, which might largely suppress the proliferation of the invading pathogen (Fig. 4D). Conversely, we observed dense intercellular hyphae with round haustoria and sporulation in the susceptible WT plants (Fig. 4E). In the transgenic line R2Y4 expressing NP::RPW8.2-YFP, the oomycete pathogen seemed to be stressed because there were many oospores and bulged haustoria (Fig. 4F and 4G), although
resistance gene RPP7 (a NB-LRR type R gene) (Nemri et al. 2010), we observed HR along the invading hyphae (Fig. 4H). These observations suggest that ectopic expression of RPW8.1-YFP can activate multiple layers (penetration and post-penetration) of resistance against Hpa Noco2, which appears to be different from typical R gene-mediated HR.
RPW8.1-YFP preferentially accumulates in mesophyll cells beneath the haustoria of powdery mildew To investigate how RPW8.1 activates resistance to Gc UCSC1, we examined subcellular defense responses. R1Y4 plants were inoculated with Gc UCSC1 and the accumulation of RPW8.1-YFP was observed by Laser Scanning Confocal Microscopy (LSCM). As shown in Fig. 5, compared to that in adjacent mesophyll cells, RPW8.1-YFP accumulated at higher levels in ring structures surrounding chloroplasts in the mesophyll cells beneath the epidermal cell in which a haustorium was developed. We also assessed the frequencies for the formation of an encasement of the haustorial complex (EHC) or HR that were associated with haustorial development at 5 dpi by trypan blue staining. Results with statistic analysis were summarized in Fig. 6A. Generally there were three types of post-invasion consequences: (i) formation of a healthy haustorium (i.e.
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R2Y4 was fully susceptible. In the accession Ms-0 that may contain a typical downy mildew
without an EHC); (ii) formation of an encased haustorium (i.e. with an EHC); and (iii) activation of HR cell death as shown in Fig. 6B-6D. The EHC was enriched in callose as detected by aniline blue staining (Fig. 6B). Previously, we found that both the EHC formation and HR contribute to post-invasion resistance and the well-adapted powdery mildew isolate Gc UCSC1 can actively suppress this defense mechanism (Wang et al. 2009; Wen et al. 2011). Consistent with the susceptible phenotype, most (91.5%) haustoria formed in the control line (C15) transgenic for 35S::YFP were healthy, only 6% haustoria were encased, and 2.5% haustorial invasion triggered
resistant lines harboring either one or both of RPW8.1 and RPW8.2 (Fig. 6A). Notably, the type (ii) in R1Y4 plants accounted for 17.9%, which was significantly higher than that in R2Y4; while the type (iii) in R1Y14 accounted for 29.9%, which was significantly lower than that in R2Y4 (Fig. 6A). Nevertheless, R1Y6 plants, which were fully susceptible to Gc UCSC1 (Fig. 3C), showed similar frequencies for the incompatible types (i.e. types ii and iii) as C15 plants, presumably due to the relatively low level of RPW8.1-YFP expression (Fig. 6A, Supplementary Fig. S1). These data suggest that RPW8.1 is able to activate effective resistance against powdery mildew when sufficiently expressed, even though it is less potent in triggering cell death in response to the haustorial invasion compared to RPW8.2. Because the formation of the EHC or HR is usually preceded by H2O2 production, we examined whether RPW8.1-mediated resistance correlates with H2O2 accumulation. When the EHC was present, H2O2 was restricted at the penetration site and contained in the haustorial complex by the EHC (Fig. 6E-6G). In the case of cell death, we observed that H2O2 was at a higher level in the dying epidermal cell, while the intercellular area beneath also contained a high concentration of H2O2 (Fig. 6H-6I). In both cases, we observed small vesicles containing H2O2 in the intercellular space and higher amount of H2O2 at the border of mesophyll cells beneath the 11
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HR (Fig. 6A). In contrast, the frequencies of the defense types were significantly higher in the
infection site (Fig. 6G, 6I). Given that chloroplasts are one major source of pathogen-induced H2O2 production and RPW8.1 is specifically localized in the periphery of chloroplasts, our observations imply that RPW8.1 may exploit chloroplasts for H2O2 production and that there may be H2O2 transport from mesophyll cells to the intercellular area and neighboring epidermal cells. Alternatively, the expression of RPW8.1 may activate plasma membrane-associated apoplastic H2O2 production. However, further investigation is necessary to clarify how H2O2 production is initiated by the expression of RPW8.1.
The mesophyll-cell-accumulation of RPW8.1-YFP expressed by its native promoter implies that the RPW8.1 promoter may be activated differently compared to the RPW8.2 promoter. To test this hypothesis, we made reporter genes expressing YFP under the control of the RPW8.1 or RPW8.2 promoter (i.e. PRPW8.1::YFP and PRPW8.2::YFP), respectively. The reporter genes were introduced into Col-gl by agro-mediated floral dipping (Clough and Bent 1998). Then the YFP expression pattern was examined under an epi-fluorescence microscope or LSCM from 2 dpi to 4 dpi of Gc UCSC1 on five-week old plants. YFP expressed from the RPW8.1 promoter was observed in both infected and uninfected cells with more intense signal in some cell clusters associated with fungal hyphae (Fig. 7A). Under higher magnification, it was clear that the YFP signal in both epidermal cells and mesophyll cells associated with fungal hyphae was stronger than that in the uninfected area (Fig. 7B). In contrast, YFP expressed from the RPW8.2 promoter was almost exclusively detected in cells at the infection site (Fig. 7C). Under higher magnification, it was clear that YFP was accumulated at high levels specifically in 1-3 epidermal cells at the infection site and in the mesophyll cell beneath the invading haustorium (Fig. 7D). These observations indicate that while the RPW8.2 promoter is specifically activated in cells at the
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Activity of the RPW8.1 promoter is distinct from that of the RPW8.2 promoter
infection site, the RPW8.1 promoter is constitutively active.
RPW8.1-YFP localizes at the EHM when expressed by the RPW8.2 promoter RPW8.1-YFP expressed by its native promoter may not sufficiently accumulate in epidermal cells, providing a plausible explanation for its lack of EHM-localization (Wang et al., 2009; this study), even though it could activate effective resistance to both virulent oomycete and powdery mildew pathogens. We thus questioned if RPW8.1-YFP could also be targeted to the EHM when
induced by downy mildew in mesophyll cells (data not shown). To this end, we constructed transgenic Col-gl plants expressing RPW8.1-YFP by the RPW8.2 promoter (i.e. PRPW8.2::RPW8.1YFP). After inoculation of Gc UCSC1, RPW8.1-YFP was clearly detectable at the EHM encasing the haustorial complex in epidermal cells (Fig. 7E), indicating that RPW8.1 possesses the capability for localization at the EHM induced by powdery mildew similar to that of RPW8.2 (Wang et al. 2009). Meanwhile, in the mesophyll cells of the same transgenic plant, RPW8.1-YFP was also observed in the periphery of chloroplasts, especially those that aggregated possibly as a result of RPW8.1-YFP accumulation (Fig. 7F). In contrast, when expressed by the RPW8.1 promoter, RPW8.2-YFP was rarely detectable in epidermal or mesophyll cells. In such rare cases, the RPW8.2-YFP signal was found as punctate spots on or peripheral to the EHM (Fig. 7G), similar to the localization pattern of RPW8.2-YFP expressed from the 35S promoter (Fig. 2G in Wang et al. 2010). Then we questioned if the Col-gl plants transgenic for PRPW8.2::RPW8.1-YFP exhibited disease resistance to powdery mildew and downy mildew. We first examined resistance to powdery mildew by inoculation of Gc UCSC1 on 5-week old plants and recorded the phenotypes at 8 dpi. As expected, these plants were all resistant to powdery mildew (Fig. 8A). The resistance
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sufficiently accumulated in epidermal cells, although it did not show localization at the EHM
phenotype was associated with fungus-induced cell death as detected by trypan blue staining (Fig. 8B) and the cell death was accompanied with H2O2 production (Fig. 8C). In addition, these lines also showed resistance to the oomycete pathogen Hpa Noco2 as indicated by the infection phenotypes at 6 dpi (Fig. 8D). The resistance to Hpa Noco2 was mainly due to cell death. As shown in Fig. 8E and 8F, there were dead host cells along the intercellular hyphae and the hyphae could rarely differentiate haustoria in the host cells. Only occasionally did we observe the formation of a haustorium (Fig. 8F inset).
suggest that appropriate ectopic expression of RPW8.1 alone may be used to engineer resistance against fungal and oomycete pathogens.
Discussion Functional diversification among members of the RPW8 family Resistance genes are often located tandemly in a genome. The RPW8 locus in A. thaliana accession Ms-0 consists of two resistance genes, RPW8.1 and RPW8.2, and three paralogs, HR1 (Homolog of RPW8 1), HR2 and HR3 (Xiao et al. 2001; Xiao et al. 2004). This locus is conserved in Brassicaceae, though with some variation. In most A. thaliana accessions, the RPW8 locus contains these five genes; whereas in some accessions, such as Col-0, RPW8.1 and RPW8.2 are replaced by HR4, a paralog showing relatively higher homology to RPW8.1 than to RPW8.2 (Orgil et al. 2007; Xiao et al. 2004). HR1, HR2 and HR3 do not make significant contribution to resistance against powdery mildew or downy mildew as indicated by the susceptibility of Col-0 to these pathogens (Xiao et al. 2001; Xiao et al. 2004). In A. lyrata, which is a close relative of A. thaliana, the RPW8 locus contains four genes, HR1, HR2, HR3 and RPW8.2, in which RPW8.2 appears to contribute strongly to resistance against powdery mildew
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Taken together, our data indicate that RPW8.1 is functionally distinct from RPW8.2 and
(Jorgensen and Emerson 2009; Orgil et al. 2007; Xiao et al. 2004). In Brassica, there are two syntenic loci: one locus contains three genes, HRa, HRb and HRc, presented in both B. rapa and B. oleracea, and the other locus contains one gene, HRd, which only exists in B. rapa (Xiao et al. 2004). Both RPW8.1 and RPW8.2 contribute to broad-spectrum resistance to mildews (Gollner et al. 2008; Jorgensen 2012; Jorgensen and Emerson 2009; Xiao et al. 2001). HR4 may be involved in the beneficial interaction between Arabidopsis and Trichoderma atroviride (Saenz-Mata and Jimenez-Bremont 2012).
genes that confer resistance against four diverse powdery mildew isolates mainly through the SAdependent signaling pathway (Xiao et al. 2003; Xiao et al. 2001). Transcription of RPW8 is regulated via a SA-dependent feedback loop and over expression of RPW8 from the native promoters can initiate spontaneous HR-like cell death in a dosage-dependent manner (Xiao et al. 2003). In addition, RPW8 is able to activate basal resistance and enhance resistance to multiple biotrophic pathogens under certain conditions (Wang et al. 2007). However, those studies used the transgenic lines expressing both RPW8.1 and RPW8.2; disease resistance of those plants was probably achieved by both RPW8.1 and RPW8.2 acting additively or synergistically. RPW8.2 alone has been shown to confer resistance to powdery mildew (Wang et al. 2010; Wang et al. 2009), how RPW8.1 alone activates resistance was not fully known. Our data in the present study suggest that ectopic expression of RPW8.1 alone is able to activate resistance against fungal (Gc UCSC1) and oomycete (Hpa Noco2) pathogens, while RPW8.2 only confers resistance to powdery mildew under the laboratory conditions described.
Functional comparison between RPW8.1 and RPW8.2
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In previous studies, RPW8.1 and RPW8.2 were characterized as broad-spectrum resistance
Using YFP as a reporter, we discovered that RPW8.2-YFP is specifically localized at the EHM whereby it initiates resistance against powdery mildew (Wang et al. 2009). In addition, 14-3-3 lambda and PAPP2C, a protein phosphatase type 2C, act as positive and negative regulators of RPW8.2, respectively (Wang et al. 2009; Wang et al. 2012; Yang et al. 2009). Given the low-tomoderate sequence identity (45%) between RPW8.1 and RPW8.2, functional diversification between these two proteins is anticipated. Indeed, through ectopic expression of YFP-tagged RPW8.1 from its native promoter or from the RPW8.2 promoter, we demonstrated that RPW8.1
difference is that cell death caused by the expression of RPW8.1 appears to be tender than that of RPW8.2. Instead of clear HR-like lesions visually visible in leaves of RPW8.2-YFP-expressing plants, plants expressing RPW8.1-YFP developed small pits on leaf surface, each of which is attributable to the collapse of a cluster of cells and contraction of surrounding cells (Fig. 1). Apparently, just like the formation of HR-like lesions in RPW8.2-YFP-expressing plants, pit development in RPW8.1-YFP-expressing plants is probably due to local accumulation of RPW8.1-YFP (Fig. 2 and Supplementary Fig. S2). Interestingly, RPW8.1-YFP expressed from the RPW8.2 promoter rarely caused pit formation (Fig. 8), which may be explained by the relatively even expression of RPW8.1-YFP from the RPW8.2 promoter in epidermal and mesophyll cells (Fig. 7). Secondly, ectopic expression of RPW8.1-YFP from either the RPW8.1 promoter or the RPW8.2 promoter confers resistance to virulent powdery mildew and oomycete pathogens, whereas RPW8.2-YFP expressed from the RPW8.2 promoter only activates resistance to powdery mildew. In addition, RPW8.2-YFP seemed unstable when expressed from the RPW8.1 promoter (Fig. 7) and could not accumulate to a level sufficient to render resistance to any pathogens (data not shown). These results reflect the functional difference between RPW8.1 and
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and RPW8.2 have distinct functions in cell death and defense activation. The first noticeable
RPW8.2, and suggest that RPW8.1 is probably more potent in defense activation although it activates milder cell death. Our observations highlight the importance of spatiotemporal expression in specifying the defense function of these two proteins. The resistance levels did not seem to strictly correlate with the expression levels of RPW8.1-YFP (Fig. 3, Supplementary Fig. S1). This observation is consistent with a previous report that the expression levels of RPW8 varied in different backgrounds but did not tightly correlate with the levels of resistance against powdery mildew
agrees well with its predominant mesophyll cell expression, since this oomycete pathogen invades both epidermal and mesophyll cells with majority of its haustoria being formed in mesophyll cells. We noted that based on promoter::YFP assays, the RPW8.1 promoter seemed to be active in epidermal cells albeit at a lower level compared with that in mesophyll cells (Fig. 7); however, RPW8.1-YFP hardly accumulated in epidermal cells when expressed by its native promoter (Fig. 2 and Supplementary Fig. S2). One possibility is that chloroplast-association may somehow enhance stable accumulation of RPW8.1-YFP, and because there are just a few plastids in epidermal cells, RPW8.1-YFP may be less stable there. The instability of RPW8.1-YFP was supported by the detection of a band at YFP size in western blot analysis (Supplementary Fig. S1B). Nevertheless, RPW8.1 may still function in epidermal cells because some Col-gl lines ectopically expressing RPW8.1-YFP also conferred resistance to powdery mildew (Fig. 3 and 8), which is an epidermal cell-limited pathogen. This speculation is supported by EHM-localization of RPW8.1-YFP when it was expressed from the RPW8.2 promoter. These observations suggest that RPW8.1 and RPW8.2 may possess similar protein-sorting signal(s) required for EHM-specific localization in the epidermal cells invaded by powdery mildew.
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(Jorgensen 2012). Nevertheless, the capability of RPW8.1 in conferring resistance to Ha Noco2
Although RPW8.1-YFP was also targeted to the EHM in epidermal cells invaded by haustoria of powdery mildew, we never reliably detected RPW8.1-YFP at the EHM induced by Hpa Noco2 in mesophyll cells. This suggests that the EHM induced by oomycete pathogens in mesophyll cells is probably different from that induced by powdery mildew in epidermal cells. This speculation is compatible with our earlier observation that RPW8.2-YFP was only found as punctate spots at or peripheral to the EHM induced by Hpa Noco2 (Wang et al., 2009). It is interesting to note that a number of plasma membrane proteins such as FLS2 and PEN1 were
Phytophthora infestans in N. benthamiana (Lu et al. 2012), whereas PEN1 was not found at the EHM induced by powdery mildew in epidermal cells (Wang et al, 2009). These published results also support the notion that the EHM induced by different haustorium-forming pathogens and/or formed in different types of host cells may be distinct in lipid and protein composition.
Possible synergistic and additive roles of RPW8.1 and RPW8.2 in defense signaling The distinct spatial expression patterns of RPW8.1 and RPW8.2 suggest that these two genes may co-operate in conferring more effective resistance to powdery mildew. It is conceivable that the epidermal cell-localized RPW8.2 is the first responder to powdery mildew infection: RPW8.2 expression is induced upon infection and targeted to the EHM to initiate onsite defense. Meanwhile, RPW8.1 is predominantly produced in the mesophyll cells underneath the attacked epidermal cells and localized to the periphery of chloroplasts. Given that RPW8.1-expression triggers H2O2 accumulation in the intercellular area of mesophyll cells and the chloroplast is one important source of H2O2 involved in defense (Chang et al. 2009; Liu et al. 2007), it is reasonable to speculate that RPW8.1 may exploit chloroplasts or the plasma membrane-localized oxidases for rapid production of apoplastic H2O2, which may then be delivered to the neighboring epidermal 18
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reported to localize at the EHM induced by Hpa Noco2 in Arabidopsis and/or the EHM induced by
cells (particularly at the host-pathogen interface) for haustorium killing or remain in the intercellular area for orchestration of defense signaling in cells surrounding the penetrated epidermal cells. Indeed, RPW8.1-YFP is specifically induced at higher levels in the mesophyll cells underneath mildew-infected epidermal cells (Fig. 5), supporting the above speculation. Our observations also agree with an earlier report that spontaneous HR-like cell death caused by the expression of RPW8.1 and RPW8.2 from their native promoters occurs first in mesophyll cells (Xiao et al., 2003). Therefore, it is apparent that both the coding sequences and promoter regions
duplication of an RPW8.2-like progenitor gene (Xiao et al., 2004), the sequence diversification and functional specialization between RPW8.1 and RPW8.2 probably reflect natural selection driven by multiple types of pathogens. How RPW8.1 might positively regulate H2O2 production and distribution in the intercellular area of mesophyll cells is an interesting question for future studies. The formation of the EHC is a highly conserved subcellular defense response against the invasion of powdery mildew. It is reported that the EHC is enriched in callose and plant cell wall polymers, such as β-linked galactose, xyloglucan, rhamnogalacturonan I, and arabino-galactan proteins (Meyer et al. 2009; Micali et al. 2011). Gc UCSC1 is a well-adapted powdery mildew pathogen, while G. orontii and Gc UMSG1 are adapted and poorly-adapted pathogens to Arabidopsis, respectively; EHC accounted for about 5% of Gc UCSC1 haustoria in Col-0 leaves, but increased to about 20% of G. orontii and more than 40% of Gc UMSG1 haustoria, respectively (Meyer et al. 2009; Wen et al. 2011), implying that the well-adapted Gc UCSC1 can suppress the defense response more effectively than G. oronti and Gc UMSG1. RPW8.1 appears to be able to counter the suppression of the EHC formation by the fungal pathogens, explaining in part the capacity of RPW8.1 in enhancing basal resistance (Fig. 6). This is consistent with a
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of these two R genes show functional diversification. Since RPW8.1 probably originated from
previous report that RPW8 (RPW8.1 and RPW8.2) is able to recover the super-susceptible phenotypes of the SA-signaling mutants to the level comparable with WT, though the full function of RPW8-mediated resistance to powdery mildew requires components of SA-signaling (Xiao et al. 2005). Taken together, our results demonstrate that RPW8.1 is distinct from RPW8.2 in terms of spatiotemporal expression pattern, protein subcellular localization, and efficacy of defense activation, thereby supporting the notion that RPW8.1 and RPW8.2 could function independently,
Materials and methods Plant materials and growth conditions Transgenic Col-gl (accession Col-0 containing the glabrous mutation, gl-1) lines expressing RPW8.2-YFP (R2Y4) and RPW8.1-YFP (numbered from R1Y1 to R1Y6) from the respective native promoters were from previous studies (Wang et al. 2007; Wang et al. 2009). Arabidopsis accession Ms-0 from which RPW8.1 and RPW8.2 were originally isolated and/or a homozygous Col-gl transgenic line S5 harboring a single copy of RPW8.1 and RPW8.2 under control of the native promoters (Xiao et al. 2003) were used as resistant references and a homozygous Col-gl transgenic line C15 expressing YFP under control of the 35S promoter (Wang et al. 2007) as susceptible reference in the pathogenesis tests. Seeds were sown in Sunshine Mix #1(Maryland Plant and Suppliers, Baltimore, MD, USA) and cold treated (4ºC for 2 d), and seedlings were kept 2
1
under 22ºC, 75% relative humidity, short-day (8 h light at 125 µmol m- s- , 16 h dark) conditions for 2 weeks and 5–6 weeks before downy mildew and powdery mildew pathogen inoculation, respectively.
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additively, and probably cooperatively in natural Arabidopsis accessions.
DNA constructs and plant transformation Plasmids for NP::RPW8.1-YFP and NP::RPW8.2-YFP were made in a previous report (Wang et al. 2007). As for promoter reporters, the RPW8.2 native promoter was amplified with primers EcoR82PF (5’-CAG AAT TCA CCG AAA TTG TTA GTA TTC A-3’) and BamR82PR (5’-ATG GAT CCG AAA TTA GTT TGT TAG CTC TCG AG-3’), digested with EcoRI and BamHI, and cloned into pPZP211 (Hajdukiewicz et al. 1994) EcoRI/BamHI site, generating an intermediate vector pPR8R5. Then, the cassette containing eYFP and RPW8.2 3’-UTR was amplified with primers
and cloned into pPR8R5 BamHI site, generating the binary vector pP2Y3’ as the RPW8.2 promoter reporter. The RPW8.1 native promoter was amplified with primers EcoR81PF1 (5’-CCG AAT TCT GCC ACA TTG GTC TCT CA-3’) and BamR81PR1 (5’- AAG GAT CCA AAG TAG TTG TTT AGC TCT CGA GG-3’), digested with EcoRI and BamHI, and cloned into pPZPYFP13′ (Wang et al. 2007) EcoRI/BamHI site, generating the binary vector pPR81Y1 as the RPW8.1 promoter reporter. As for expressing RPW8.1-YFP from the RPW8.2 promoter, the coding region of RPW8.1 was amplified with primers BMC2F (5’-CAG GAT CCA TGC CGA TTG GTG AGC TTG CGA TA-3’) and BamR81R (5’- CGC GGA TCC AGC TCT TAT TTT ACT ACA AGC AGA-3’), digested with BamHI and cloned into pP2Y3’ BamHI site, resulting in the plasmid P2R81Y7 (PRPW8.2::RPW8.1-YFP). As for expressing RPW8.2-YFP from the RPW8.1 promoter, the coding region of RPW8.2 was amplified with primers BamR82F (5’-CAG GAT CCA TGA TTG CTG AGG TTG CCG CA-3’) and BamR82R (5’- CGC GGA TCC AGA ATC ATC ACT GCA GAA CGT AAA3’), digested with BamHI and cloned into pPR81Y1 BamHI site, resulting in the plasmid P1R82Y2 (PRPW8.1::RPW8.2-YFP). The constructs were individually introduced into Col-gl via Agro-mediated floral dipping (Clough and Bent 1998).
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BamYFPF1 and BglR823’R from pPR82EYFP (Wang et al. 2007), digested with BamHI and BglII,
Pathogen infection and microscopy analyses The powdery mildew isolate Gc UCSC1 was maintained on live eds1-2 or pad4-1 plants for generation of fresh inoculums. The fresh inoculums of downy mildew isolate Hpa Noco2 was prepared on eds1-2 leaves by inoculating conidia stored in a -80 freezer. Inoculation, scoring of mildew disease reaction phenotypes and quantification of disease susceptibility were done as previously described (Wang et al. 2007; Xiao et al. 2003). Dead cells and fungal structures in
mildew and 6 dpi for downy mildew. Statistic analysis on Gc UCSC1 haustorial types were followed the previous study (Wang et al. 2009). Examination of H2O2 in situ production was conducted as in Xiao et al. (2003). In situ detection of callose was performed as previously described (Wang et al. 2009). Laser scanning confocal microscopy (LSCM) images were acquired as previously described (Wang et al. 2007) by using Zeiss LSM 710 microscope or following the user’s manual by using Nikon A1 microscope. All pictures presented in the figures were projections from Z-stacks of 15 to 60 images unless otherwise indicated. The image data were processed using Zeiss LSM Image Browser or NIS-Elements viewer and Adobe Photoshop. Transcript and protein analysis RNA extraction and quantification of the transcripts of RPW8.1 and ACT2 by real-time PCR was performed as described by Xiao et al. (2003). PCR was performed using an ABI 7300 sequence detection system (Applied Biosystems). All reactions were performed in triplicate. Protein extraction and gel blot analysis were performed as previous report (Wang et al. 2007) with antiGFP serum that could also detect YFP.
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inoculated leaves were examined with trypan blue staining (Xiao et al. 2003) at 5 dpi for powdery
Funding This work was supported by National Natural Science Foundation of China (grant numbers 31071670 and 31371931 to W.-M.W.); Sichuan Research Program of International Cooperation and Exchanges (grant number 2012HH0012 to W.-M.W.); and by National Science Foundation (grant numbers IOS-0842877, IOS-1146589 to S.-Y.X.).
Acknowledgments
for the H. arabidopsidis Noco2 isolate, Xinnan Wang for critical reading of the manuscript.
References
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Figure Legends Fig. 1. The expression of RPW8.1-YFP leads to spontaneous cell death distinguishable to those caused by the expression of RPW8.2-YFP. (A) A representative five-week-old plant from the indicated lines showing the phenotypes of Col-gl (wild type) expressing YFP from the 35S promoter (C15) and three transgenic lines (R1Y1, R1Y4 and R1Y6) expressing RPW8.1-YFP by the native promoter. (B) A representative leaf from 5-week-old R1Y4 plant showing some pits (arrows) on the adaxial side of the leaf. (C) The same leaf of B showing the bulges (arrows) on
(arrows) in R1Y4 with necrotic lesions (arrow heads) in R2Y4. (E-F) A representative section of a trypan blue-stained leaf from a 6-week-old R1Y4 plant showing a cluster of dead cells centered by radially arranged cells (E) and a cluster of dead cells surrounded by irregular-shaped epidermal cells (F). Note that there was a cluster of small mesophyll cells below the dead epidermal cells. (G) A representative section of a leaf from R2Y4 showing a cluster of dead epidermal cells (stars) and mesophyll cells (arrows). Note there was no shape change in cells around the dead cells. (H-I) A representative section of a leaf from R1Y4 (H) and R2Y4 (I) stained by DAB showing that H2O2 was accumulated in the intercellular area (arrows) and surrounded by oval-shaped mesophyll cells (*) in R1Y4, but H2O2 was accumulated inside a mesophyll cell and there was no cell-shape change in the surrounding mesophyll cells in R2Y4. Bars, 50 µm in E-G, 20 µm in H-I.
Fig. 2. Subcellular localization and accumulation patterns of RPW8.1-YFP from its native promoter (NP::RPW8.1-YFP). Representative confocal images acquired from leaves of 5-week old plants. Yellow fluorescent protein (YFP) tagged RPW8.1 protein was pseudocolored green, whereas the autofluorescent chloroplasts and propidium iodide (PI) stained plant structures were
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the abaxial side of the pits. (D) Representative leaves from the indicated lines for comparing pits
pseudocolored blue and red, respectively. Bars, 10 µm. (A) RPW8.1-YFP highly accumulated in a cluster of mesophyll cells (dotted-line area) and the protein was located surrounding chloroplasts. (B) RPW8.1-YFP highly accumulated in an area (indicated by dotted-line) where cell shape changes occurred in the adjacent cells (*) as revealed by PI staining.
Fig. 3. The expression of RPW8.1-YFP was sufficient to render resistance against powdery mildew and downy mildew. For downy mildew infection, two-week-old seedlings were inoculated
6 dpi. For powdery mildew infection, 5-week-old plants were inoculated with Gc UCSC1 and infection results were recorded at 8 dpi. (A-B) Representative leaves from the indicated transgenic Col-gl lines expressing YFP (C15) from 35S or RPW8.1-YFP (R1Y1-R1Y6) from the RPW8.1 native promoter showing the disease phenotypes of downy mildew at 6 dpi (A) and powdery mildew at 8 dpi (B). Note that the lines R1Y1, R1Y4 and R1Y5 exhibited enhanced resistance to both mildews, whereas, R1Y6 was as susceptible to powdery mildew as C15 but displayed enhanced resistance to downy mildew. (C) Downy mildew susceptible phenotypes for the indicated lines at 6 dpi. S5 was Col-gl transgenic line harboring a single copy of RPW8 (RPW8.1 and RPW8.2) under control of the native promoters (Xiao et al. 2003). R2Y4 was a Colgl transgenic line expressing RPW8.2-YFP from its native promoter (Wang et al. 2009). (D) Powdery mildew resistant phenotypes for the indicated lines with C15 being a susceptible reference at 8 dpi. (E-F) Quantitative assay of disease susceptibility to Hpa Noco2 at 6 dpi (E) and Gc UCSC1 at 7dpi (F), respectively. Values are means of three replications. Error bars indicate SD. Student’s t test was carried out to determine the significance of differences between C15 and the indicated lines. Asterisks indicate significant differences at P200 interaction sites were evaluated). Asterisks (* and **) indicate significant difference from C15 at a P value