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Hemibiotrophs, such as Phytophthora infestans, exhibit distinct phases of their life cycle: an early asymptomatic biotrophic phase and a late necrotrophic stage that is characterized by tissue degradation and disease symptoms. To date, little is known of the molecular mechanisms that promote each distinct phase, nor those that mediate the transition between the two. We hypothesized that these phytopathogens might secrete distinct classes of effector proteins that first suppress plant defense responses and associated programmed cell death (PCD), and later induce large scale necrosis. To this end, we have identified proteins that are secreted by P. infestans early or late in the infection cycle. Recently we described the characterization of SNE1, which is specifically expressed during early biotrophic growth in the host plant tomato (Solanum lycopersicum). We found that SNE1 suppresses the action of necrosis-inducing effectors (Nep1-like proteins), including PiNPP1.1 and PsojNIP, which are secreted by P. infestans and P. sojae, respectively, during necrotrophic growth, as well as PCD mediated by a broad spectrum of Avr-R protein interactions. This suggests that SNE1 and PiNPP1.1 act antagonistically, thereby providing a highly regulated means to control the transition from biotrophy to necrotrophy.
A spectrum of hemibiotrophic plant pathogens, including the bacterium Pseudomonas syringae, the fungus Colletotrichum graminicola and the oomycete Phytophthora infestans, exhibit characteristics of both biotrophs and necrotrophs, depending on the stages of their life cycles. In the early stages of infection, the pathogens proliferate asymptomatically in the host by suppressing programmed cell death (PCD) or thwarting host defense responses, but in the later stages of infection they undergo a physiological transition from asymptomatic biotrophic growth to a highly destructive necrotrophic phase.1 Hemibiotrophic bacteria are known to secrete a range of so-called effector proteins, including transcription factors and others with enzymatic activities, into host cells via the type III secretion system (T3SS),2 whereupon they suppress PCD and other host defenses.3,4 However, far less is understood about equivalent systems in other eukaryotic plant pathogens. The T3SS is not present in fungi and oomycetes, which are thought to introduce proteins in the host by trafficking through their own canonical endoplasmic reticulum-Golgi secretory pathway and subsequent translocation into the host cell, but this molecular machinery involved in protein translocation is unknown.5–7
In addition, the biological roles and modes of action of most secreted effector proteins from fungal and oomyceteous pathogens are still poorly defined, although some effectors have been demonstrated to suppress PCD or host defenses. For example, the potato late blight pathogen P. infestans effector Avr3a has been shown to inhibit cell death triggered by the INF1 elicitor, a cell death inducing protein from P. infestans.8 Similarly, the soybean pathogen Phytophthora sojae effector Avr1b can suppress PCD triggered by the mammalian BAX protein, which is a positive regulator of apoptosis.9 These results imply that these secreted effectors play an important role in suppressing PCD or host defenses during biotrophic interactions.10,11 However, it is still not clear how secreted effector proteins might modulate plant defense and surveillance systems more broadly to control the transition from biotrophy to necrotrophy.
In a recently published paper,12 we described the identification and characterization of a secreted effector (SNE1) from P. infestans that encodes a highly hydrophilic protein with a predicted secretory signal peptide (SP) and nuclear localization signals (NLSs). SNE1 suppresses PCD induced by divergent molecular pathways and is specifically expressed during early biotrophic growth, while conversely, the P. infestans PiNPP1.1 gene, which encodes a member of the class of secreted Nep1-like proteins (NLPs) that induce host cell death,13 is highly expressed during necrotrophy. Thus, SNE1 and PiNPP1.1 are coordinately expressed during biotrophy and necrotrophy, respectively.12 We therefore hypothesized that SNE1 blocks the destructive action of PiNPP1.1 to maintain a biotrophic phase before the transition to necrotrophy is triggered.
To test our hypothesis, SNE1 plus one of two different NLPs (PiNPP1.113 from P. infestans and PsojNIP14 from P. sojae) were transiently co-expressed using agroinfiltration in leaves of Nicotiana benthamiana and tomato. We thereby showed that indeed, SNE1 suppresses the PCD induced by both NLPs in both solanaceous species.12 We then went on to demonstrate that SNE1 also inhibits PCD initiated by the Avr-R protein interactions from a broad spectrum of pathosystems, including oomycetes (Avr3a/R3a), bacteria (AvrPto/Pto), viruses (CP/R×2) and fungi (Avr9/Cf9).12 SNE1 is therefore a secreted effector protein with a remarkably broad-spectrum suppressive activity; a result that, together with the expression pattern, supports our hypothesis that hemibiotrophic pathogens secrete antagonistic effector proteins during two distinct phases of hemibiotrophy. Thus, P. infestans appears to have evolved an elegant coordinated system to control the transition from biotrophy to necrotrophy (Fig. 1); in effect using a simultaneous ‘accelerator and brake’ strategy to regulate the onset of necrosis and disease symptoms. We further suggest that similar regulatory systems are likely to be present in other oomycetes, and potentially other eukaryotic plant pathogens.
As mentioned, very little is known about the translocation machinery of fungal and oomyceteous effector proteins. Intriguingly, many oomycete effector proteins have the N-terminal motif RXLR-dEER,15,16 which is very similar to the motif RXLXE/Q/D of effector proteins from the malaria pathogen Plasmodium falciparum.17,18 It has been shown that the oomycete RXLR-dEER and Plasmodium RXLXE/Q/D motifs seem to function inter-changeably19–21 and that the former is necessary for the translocation of P. infestans Avr3a into infected plant cells.22 Although direct evidence that SNE1 enters the plant cells has not been presented, SNE1 contains a variant of RXLR-dEER motif (RXLX)12 that is reminiscent of the Plasmodium RXLXE/Q/D motif. We note that the bean rust secreted proteins (RTP1p) containing the RXLX motif were detected in the nucleus of infected host cells,23 suggesting that the RXLX motif in SNE1 or RTP1p might be involved in translocation into host cells. Moreover, we have also demonstrated that tdTomato-tagged SNE1 has the capacity to translocate to the nucleus following heterologous expression in the plant cells, in accordance with the presence NLS motifs.12 Interestingly, it has recently been reported that the RXLXE/Q/D motif is a protease cleavage site at which the protein is cleaved immediately after the leucine (L) residue of RXLXE/Q/D in the parasite endoplasmic reticulum (ER).24–28 This obviously contradicts that idea that this peptide sequence functions as part of the protein translocation system in the host cell, and it will be important to determine whether or not the oomycete RXLR-dEER motif is similarly subject to proteolytic cleavage before leaving the oomycete secretory pathway.
Large numbers of putative new effector proteins from hemibiotrophs are now being identified from genome-scale analyses.15,16 The characterization of SNE1 and other secreted effectors, and the unraveling of the corresponding spatio-temporal order of gene expression, are filling in some missing links in our understanding of how the transition from biotrophy to necrotrophy is regulated. However, clearly there remains a great deal to be learnt about not only their modes of action, but also the mechanisms by which they arrive at the sites of action. The next crucial step will be the transition from descriptive to mechanistic information.
We thank Kent Loeffler (Cornell University) for photographic assistance. This work was supported by a grant from the NSF Plant Genome Research Program (DBI-0606595) and the New York State Office of Science, Technology and Academic Research (NYSTAR).
Addendum to: Kelley BS, Lee S-J, Damasceno CMB, Chakravarthy S, Kim B-D, Martin GB, Rose JKC. A secreted effector protein (SNE1) from Phytophthora infestans is a broadly acting suppressor of programmed cell deathPlant J2010In press doi: 10.1111/j.1365-313X.2010.04160.x.
Previously published online: www.landesbioscience.com/journals/psb/article/11778