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Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans.
Phytophthora species are among the most devastating crop pathogens worldwide. P. infestans is a pathogen of tomato and potato plants. The genome of P. infestans has been sequenced, revealing the presence of a large number of host-targeting RXLR effector proteins that are thought to manipulate cellular activities to the benefit of the pathogen. One step toward disease management comprises understanding the molecular basis of host susceptibility. In this paper, we used a protoplast-based system to analyze a subset of P. infestans RXLR (PiRXLR) effectors that interfere with plant immunity initiated by the recognition of microbial patterns (MAMP-triggered immunity - MTI). We identified PiRXLR effectors that suppress different stages early in the signaling cascade leading to MTI in tomato. By conducting a comparative functional analysis, we found that some of these effectors attenuate early MTI signaling in Arabidopsis, a plant that is not colonized by P. infestans. The PiRXLR effectors localize to different sub-cellular compartments, consistent with their ability to suppress different steps of the MTI signaling pathway. We conclude that the effector complement of P. infestans contains functional redundancy in the context of suppressing early signal transduction and gene activation associated with plant immunity.
Transient expression in protoplasts has proven fast and reliable for studying the function of bacterial type III effectors that suppress early MAMP signaling , . Moreover, the assay allows the measurement of synchronized responses and it does not require the use of bacteria for protein or DNA transfer into the host cell. In addition, the protoplast system offers the possibility to test large sets of effectors in a medium-high throughput manner. In this study, we have used tomato mesophyll protoplasts to screen a library of 33 P. infestans RXLR effector candidates (PiRXLRs) for their ability to suppress flg22-triggered defense signaling. Our additional aim was to test whether PiRXLRs that suppress early MTI signaling in the host plant tomato retain that ability in the distantly-related non-host plant Arabidopsis. For the experimental read-out we measured the abilities of these effectors to suppress: i) flg22-induced promoterFLG22-INDUCED RECEPTOR-LIKE KINASE 1 - LUCIFERASE (pFRK1-Luc) reporter gene activity; ii) flg22-induced post-translational MAP kinase activation; and iii) flg22-induced gene expression. In addition, we performed sub-cellular localization studies of fluorescent protein-tagged PiRXLR effectors by confocal microscopy. Finally, we tested the potential of the PiRXLR effectors suppressing early MTI signaling to enhance N. benthamiana susceptibility to P. infestans. Unraveling the mode-of-action of PiRXLR effectors within plant cells will help to gain insight into the specific mechanisms that coordinate different signaling and metabolic pathways to ensure proper plant development and response to environmental changes or stresses.
The sub-cellular localizations of the 3 PiRXLR effectors (SFI1, SFI2 and SFI8/AVRblb2) affecting pFRK1-Luc/MAMP gene activation in both tomato and Arabidopsis are similar in each plant species (Figure 3A). GFP-SFI8/AVRblb2 showed nuclear-cytoplasmic localization whereas GFP-SFI1 and GFP-SFI2 localized predominantly in the nucleus, and were also apparent in the nucleolus (Figure 3A). In the case of GFP-SFI1, additional fluorescence signal was observed in the cytoplasm (and possibly at the plasma membrane [PM]) (Figure 3A). The 5 PiRXLR effectors (GFP-SFI3, -SFI4, -SFI5, -SFI6 and -SFI7) with a tomato-specific effect showed different subcellular localizations. GFP-SFI3 was enriched in the nucleus/nucleolus, GFP-SFI4 showed nuclear-cytoplasmic localization, and GFP-SFI5, -SFI6 and -SFI7 showed differing degrees of cytoplasmic localization and association with the PM (Figure 3B), with GFP-SFI5 almost exclusively localized to the PM.
In tomato, 3 effectors (SFI5-SFI7) consistently suppressed flg22-dependent post-translational MAP kinase activation (Figure 4A). We confirmed this result by performing transient expression of HA-tagged SlMPK1 and SlMPK3 in protoplasts followed by immunoprecipitation and in vitro MAP kinase assay (Figure 4B). In contrast, none of the 8 SFI effectors attenuated flg22-dependent post-translational MAP kinase activation in Arabidopsis (Figure 4C). This suggests that the effectors (SFI1, SFI2 and SFI8/AVRblb2) that were shown to attenuate flg22-induced gene activation in both tomato and Arabidopsis are most likely doing so downstream of MAP kinase activation. In the case of SFI5, the demonstration that it attenuates MAP kinase activation only in tomato (Figure 4A, 4C) is consistent with the observation that, although this effector suppressed pFRK1-Luc activation in Arabidopsis, it failed to suppress flg22-mediated up-regulation of endogenous FRK1 in that plant.
In tomato and other solanaceous plants, MAP kinase signaling cascades are best studied in the context of programmed cell death (PCD) associated with effector-triggered immunity , , . In N. benthamiana, PCD triggered by perception of the P. infestans MAMP INF1 requires NbMKK1 and its interaction with SIPK (salicylic acid-induced protein kinase; an ortholog of SlMPK1) . The role of MAPKK kinases in tomato immunity is only documented for SlMAP3Kα and SlMAP3Kε ,  and the best characterized MAPK kinases are SlMEK1 and SlMEK2 . Whether these kinases contribute to flg22-triggered signaling in tomato is unknown. As shown in Figure S10, transient expression in tomato protoplasts of a constitutively active SlMEK2 (SlMEK2-DD), or the kinase domain of SlMAP3Kα (SlMAP3Kα-KD), led to post-translational activation of SlMPK1 and SlMPK3 in the absence of flg22. The constitutively active SlMEK1 (SlMEK1-DD) and kinase domain of SlMAP3Kε (SlMAP3Kε-KD) did not activate SlMPK1 and SlMPK3. The expression of the constitutively active SlMEK2 (SlMEK2-DD) and the kinase domain of SlMAP3Kα (SlMAP3Kα-KD) overrode the suppression of flg22-dependent activation of SlMPK1 and SlMPK3 by SFI5-SFI7 (Figure 5A, 5B). These results indicate that the three effectors suppress the signaling cascade very early; either upstream of MAPKK kinase activation, or specifically at the MAPK- and/or MAPKK kinase(s) involved in flg22 signaling. This is consistent with association of these effectors with the plant plasma membrane, where they may interfere with the earliest components of MAMP perception or signal transduction.
In this study, we used a protoplast-based system to assess the potential for RXLR effectors from P. infestans (PiRXLRs) to manipulate MAMP-triggered early signaling in both a host and non-host plant species. Of 33 PiRXLR effector candidates, selected on the basis of up-regulation during the biotrophic phase of late blight infection, 8 (SFI1-SFI8) were able to suppress flg22-mediated induction of pFRK1-Luc activity in protoplasts of the host plant tomato (summarized in Table 1). Of these, three (SFI5-SFI7) were shown to suppress flg22-dependent MAP kinase activation at - or upstream of - the step of MAPK- and/or MAPKK kinase activation, indicating that they target the earliest stages of MTI signal transduction in tomato (Table 1). As P. infestans does not possess flagellin, the ability of these effectors to attenuate flg22-mediated MAP kinase activation and early defense gene expression indicates that these events are likely stimulated following perception of as yet undefined oomycete MAMPs. We confirmed that 7 of the 8 PiXRLR effectors that suppress early MTI signaling in tomato also enhance colonization by P. infestans in the host plant N. benthamiana (Table 1).
We found that 3 PiRXLR effectors (SFI1, SFI2 and SFI8/AVRblb2) suppress flg22-mediated induction of pFRK1-Luc activity in protoplasts of both the host plant tomato and the non-host plant Arabidopsis. We confirmed that suppression by all 3 effectors attenuates transcriptional activation of endogenous MAMP-induced marker genes in Arabidopsis (Table 1), indicating that some effectors may function efficiently across diverse (host and non-host) plant species. Interestingly, we found another set of 4 PiRXLR effectors that suppressed pFRK1-Luc activation only in the non-host Arabidopsis. This was a surprise, albeit the assay is potentially less sensitive in the host plant tomato. However, none of these effectors were able to prevent the activation of endogenous (Arabidopsis) MAMP-induced marker genes (Table 1). Therefore, additional experiments are necessary to determine to what extent suppression of flg22-induced post-transcriptional or translational processes may account for the activity of these effectors on the pFRK1-Luc reporter system in this plant. 2b1af7f3a8