Oomycete pathogens secrete hundreds of cytoplasmic RxLR effectors to modulate host immunity by targeting diverse plant proteins. Revealing how effectors manipulate host proteins is pivotal to understanding infection processes and to developing new strategies to control plant disease. Here we show that the Phytophthora infestans RxLR effector Pi22798 interacts in the nucleus with a potato class II knotted-like homeobox (KNOX) transcription factor, StKNOX3. Silencing the ortholog NbKNOX3 in Nicotiana benthamiana reduces host colonization by P. infestans, whereas transient and stable overexpression of StKNOX3 enhances infection. StKNOX3 forms a homodimer which is dependent on its KNOX II domain. The KNOX II domain is also essential for Pi22798 interaction and for StKNOX3 to enhance P. infestans colonization, indicating that StKNOX3 homodimerization contributes to susceptibility. However, critically, the effector Pi22798 promotes StKNOX3 homodimerization, rather than heterodimerization to another KNOX transcription factor StKNOX7. These results demonstrate that the oomycete effector Pi22798 increases pathogenicity by promoting homodimerization specifically of StKNOX3 to enhance susceptibility.
Plant pathogens deliver effectors to alter host processes. Knowledge of how effectors target and manipulate host proteins is critical to understand crop disease. Here, we show that in planta expression of the RXLR effector Pi04314 enhances leaf colonization by Phytophthora infestans via activity in the host nucleus and attenuates induction of jasmonic and salicylic acid-responsive genes. Pi04314 interacts with three host protein phosphatase 1 catalytic (PP1c) isoforms, causing their re-localization from the nucleolus to the nucleoplasm. Re-localization of PP1c-1 also occurs during infection and is dependent on an R/KVxF motif in the effector. Silencing the PP1c isoforms or overexpression of a phosphatase-dead PP1c-1 mutant attenuates infection, demonstrating that host PP1c activity is required for disease. Moreover, expression of PP1c-1mut abolishes enhanced leaf colonization mediated by in planta Pi04314 expression. We argue that PP1c isoforms are susceptibility factors forming holoenzymes with Pi04314 to promote late blight disease.
Plants are resistant to most microbial species due to nonhost resistance (NHR), providing broad-spectrum and durable immunity. However, the molecular components contributing to NHR are poorly characterised. We address the question of whether failure of pathogen effectors to manipulate nonhost plants plays a critical role in NHR. RxLR (Arg-any amino acid-Leu-Arg) effectors from two oomycete pathogens,
Summary A diverse range of plant proteases are implicated in pathogen perception and in subsequent signalling and execution of disease resistance. We demonstrate, using protease inhibitors and virus‐induced gene silencing (VIGS), that the plant papain cysteine protease cathepsin B is required for the disease resistance hypersensitive response (HR). VIGS of cathepsin B prevented programmed cell death (PCD) and compromised disease resistance induced by two distinct non‐host bacterial pathogens. It also suppressed the HR triggered by transient co‐expression of potato R3a and Phytophthora infestans Avr3a genes. However, VIGS of cathepsin B did not compromise HR following recognition of Cladosporium fulvum AVR4 by tomato Cf‐4, indicating that plant PCD can be independent of cathepsin B. The non‐host HR to Erwinia amylovora was accompanied by a transient increase in cathepsin B transcript level and enzymatic activity and induction of the HR marker gene Hsr203 . VIGS of cathepsin B significantly reduced the induction of Hsr203 following E. amylovora challenge, further demonstrating a role for this protease in PCD. Whereas cathepsin B is often relocalized from the lysosome to the cytosol during animal PCD, plant cathepsin B is secreted into the apoplast, and is activated upon secretion in the absence of pathogen challenge.
Plant pathogens deliver effectors into plant cells to suppress immunity. Whereas many effectors inactivate positive immune regulators, other effectors associate with negative regulators of immunity: so-called susceptibility (S) factors. Little is known about how pathogens exploit S factors to suppress immunity. Phytophthora infestans RXLR effector Pi02860 interacts with host protein NRL1, which is an S factor whose activity suppresses INF1-triggered cell death (ICD) and is required for late blight disease. We show that NRL1 interacts in yeast and in planta with a guanine nucleotide exchange factor called SWAP70. SWAP70 associates with endosomes and is a positive regulator of immunity. Virus-induced gene silencing of SWAP70 in Nicotiana benthamiana enhances P. infestans colonization and compromises ICD. In contrast, transient overexpression of SWAP70 reduces P. infestans infection and accelerates ICD. Expression of Pi02860 and NRL1, singly or in combination, results in proteasome-mediated degradation of SWAP70. Degradation of SWAP70 is prevented by silencing NRL1, or by mutation of Pi02860 to abolish its interaction with NRL1. NRL1 is a BTB-domain protein predicted to form the substrate adaptor component of a CULLIN3 ubiquitin E3 ligase. A dimerization-deficient mutant, NRL1NQ, fails to interact with SWAP70 but maintains its interaction with Pi02860. NRL1NQ acts as a dominant-negative mutant, preventing SWAP70 degradation in the presence of effector Pi02860, and reducing P. infestans infection. Critically, Pi02860 enhances the association between NRL1 and SWAP70 to promote proteasome-mediated degradation of the latter and, thus, suppress immunity. Preventing degradation of SWAP70 represents a strategy to combat late blight disease.
The potato (Solanum tuberosum) blight pathogen Phytophthora infestans delivers Arg-X-Leu-Arg (RXLR) effector proteins into host cells to subvert plant immune responses and promote colonization. We show that transient expression and stable transgenic expression of the RXLR effector Pi22926 in Nicotiana benthamiana promotes leaf colonization by P. infestans. Pi22926 suppresses cell death triggered by coexpression of the Cladosporium fulvum avirulence protein Avr4 and the tomato (Solanum lycopersicum) resistance protein Cf4. Pi22926 interacts with a potato mitogen-activated protein kinase kinase kinase, StMAP3Kβ2, in the nucleoplasm. Virus-induced gene silencing (VIGS) of the ortholog NbMAP3Kβ2 in N. benthamiana enhances P. infestans colonization and attenuates Cf4/Avr4-induced cell death, indicating that this host protein is a positive regulator of immunity. Cell death induced by Cf4/Avr4 is dependent on NbMAP3Kε and NbMAP3Kβ2, indicating that these MAP3Ks function in the same signaling pathway. VIGS of NbMAP3Kβ2 does not compromise cell death triggered by overexpression of MAP3Kε. Similarly, VIGS of NbMAP3Kε does not attenuate cell death triggered by MAP3Kβ2, demonstrating that these MAP3K proteins function in parallel. In agreement, Pi22926 or another RXLR effector, PexRD2, only suppresses cell death triggered by expression of StMAP3Kβ2 or StMAP3Kε, respectively. Our data reveal that two P. infestans effectors, PexRD2 and Pi22926, promote P. infestans colonization by targeting MAP3K proteins that act in parallel in the same signal transduction pathway.
Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses.
In nature, most plants are resistant to a wide range of phytopathogens. However, mechanisms contributing to this so-called nonhost resistance (NHR) are poorly understood. Besides constitutive defences, plants have developed two layers of inducible defence systems. Plant innate immunity relies on recognition of conserved pathogen-associated molecular patterns (PAMPs). In compatible interactions, pathogenicity effector molecules secreted by the invader can suppress host defence responses and facilitate the infection process. Additionally, plants have evolved pathogen-specific resistance mechanisms based on recognition of these effectors, which causes secondary defence responses. The current effector-driven hypothesis is that nonhost resistance in plants that are distantly related to the host plant is triggered by PAMP recognition that cannot be efficiently suppressed by the pathogen, whereas in more closely related species, nonhost recognition of effectors would play a crucial role. In this review we give an overview of current knowledge of the role of effector molecules in host and nonhost resistance and place these findings in the context of the model. We focus on examples from filamentous pathogens (fungi and oomycetes), discuss their implications for the field of plant-pathogen interactions and relevance in plant breeding strategies for development of durable resistance in crops.
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.
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