Citation: Zhang S, Xu J-R (2014) Effectors and Effector Delivery in Magnaporthe oryzae. PLoS Pathog10(1): e1003826. https://doi.org/10.1371/journal.ppat.1003826
Editor: Joseph Heitman, Duke University Medical Center, United States of America
Published: January 2, 2014
Copyright: © 2014 Zhang, Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported the National Major Project of Breeding for New Transgenic Organisms (2012ZX08009003). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Rice blast caused by Magnaporthe oryzae is one of the most destructive fungal diseases of rice and a model for studying fungal-plant interactions. The fungus penetrates plant cells with appressoria and develops the narrow primary invasive hyphae (IH) and, subsequently, the bulbous secondary IH. As a hemibiotrophic pathogen, biotrophic IH are enclosed in the extra-invasive-hyphal membrane (EIHM) produced by the plant cells . Like many other fungal pathogens , M. oryzae secretes effector proteins to manipulate plant immunity and physiology to promote infection.
Avr proteins, a special class of effectors encoded by the avirulence (AVR) genes, could be recognized by corresponding R proteins and lead to the race-specific recognition . In the rice–M. oryzae interaction, over 40 AVR genes have been identified. Among the AVR genes that have been cloned, all but ACE1 encode secreted proteins expressed in IH. ACE1 is specifically expressed in appressoria, and it encodes an intracellular hybrid PKS-NRPS protein . Avr-Piz-t and Avr-Pita are the other two Avr proteins with known biochemical functions. Avr-Piz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity by inhibiting the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 . AVR-Pita encodes a putative neutral zinc metalloprotease  and it belongs to a gene family with at least two additional members .
PWL1, PWL2, PWL3, and PWL4 are members of another AVR gene family . Pwl effectors are small, glycine-rich proteins that are present in rice pathogens and function as avirulence proteins in infection of weeping lovegrass and finger millet. In contrast, AVR1-CO39 was cloned from a weeping lovegrass isolate. Its specific expression in IH triggers hypersensitive response (HR) and resistance in cultivars carrying the Pi-CO39 R gene . Avr-Pia, Avr-Pik/km/kp, and Avr-Pii were identified in the same re-sequencing study of strain Ina168 . Avr-Pia directly interacts with Rga5-A, which also interacts with Avr-CO39 .
The best characterized non-Avr effector in M. oryzae is Slp1, a secreted LysM protein that is accumulated at the interface between IH and EIHM . Slp1 is dispensable for appressorium penetration but required for invasive growth in planta. It competes with the chitin elicitor binding protein CEBiP for binding to chitin oligosaccharides. Thus, Slp1 functions to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and expression of defense-related genes .
Using an expression profiling approach, a number of low molecular weight secreted proteins specifically expressed or highly induced in biotrophic invasive hyphae were identified, including 58 candidate effectors that were up-regulated over 10-fold during plant infection. Four of them, BAS1–BAS4, were confirmed to be fungal biotrophy-associated secreted (BAS) proteins . MC69 was identified by systematic disruption of in planta expressed secreted protein genes . The 54-aa Mc69 protein was essential for IH development, although it was not translocated into rice cytoplasm. In a separate study, five secreted protein genes named MoCDIP1–5 were found to induce plant cell death in a transient expression assay with rice protoplasts. Four of them also induced cell death in Nicotiana benthamiana .
Localization of Fungal Effectors in Plant Cells
M. oryzae effectors can be divided into two distinct types based on their localization in plant cells (Figure 1) . Cytoplasmic effectors, including Avr-Pita, Pwl1, Pwl2, Bas2, and Avr-Piz-T, are preferentially accumulated in the biotrophic interfacial complex (BIC) before being delivered into plant cells. The BIC is a distinct plant-derived, membrane-rich structure developed at the tip of primary IH by M. oryzae. In each newly invaded rice cell, effectors are first secreted into BICs before delivery. The BICs are persistent and left behind when the primary IH differentiates into the secondary IH. In addition, the fungus continues to secrete effectors into BICs even after IH have grown extensively as pseudohyphae and invaded neighboring plant cells .
A. Cytoplasmic effectors (♦) are secreted into the biotrophic interfacial complex (BIC) before being translocated into plant cells. B. Apoplastic effectors (◊) are secreted into the space between the fungal cell wall and extra-invasive-hyphal membrane (EIHM). Like IH, effector proteins may move from cell to cell via plasmodesmata. AP, appressorium.
On the other hand, apoplastic effectors such as Bas4, Avr1-CO39, and Slp1 are not associated with the BIC. After secretion, they are dispersed in the extracellular space between the fungal cell wall and EIHM. In the rice cells colonized by transformants expressing the Bas4-GFP fusion protein, GFP signals appeared to outline the IH , consistent with its extracellular localization. To date, it is not clear how the apoplastic effectors are recognized by surface receptors or translocated into plant cells to interact with their intracellular targets. Furthermore, no specific protein motifs or sequences have been identified in the cytoplasmic or apoplastic effectors that are responsible for their localization in plant cells after being secreted. Therefore, it is impossible to predict whether an effector is apoplastic or cytoplasmic solely based on its amino acid sequence.
Effector Secretion Systems
Consistent with two types of effectors, two distinct effector secretion systems have been identified in M. oryzae (Figure 2) . Cytoplasmic effectors are delivered into plant cells via the BIC, which is independent of the Golgi-dependent secretory system. Instead, secretion into the BIC is associated with a novel form of secretion involving the exocyst complex and t-SNAREs. Targeted deletions of the exocyst components SEC5 and EXO70 resulted in impaired secretion of cytoplasmic effectors and pathogenicity defects but had no effect on the secretion of apoplastic effectors . Mutants deleted of the t-SNARE component SSO1 were defective in BIC development and pathogenesis .
The apoplastic effectors (◊) utilize the conventional ER-Golgi secretion pathway for secretion. Disruption of the cytoskeleton by benzimidazoleand LatA treatments interferes with the secretion of apoplastic effectors. In contract, the secretion of cytoplasmic effectors (♦) into the BIC is independent of the Golgi bodies and cytoskeleton but involves the exocyst complex and t-SNAREs. A, cortical actin; ER, endoplasmic reticulum; EX, exocyst complex; G, Golgi bodies; M, microtubules; N, Nucleus.
The apoplastic effectors are secreted from IH by the conserved ER to Golgi secretory pathway that is independent of the BIC. Treatment with Brefeldin A that interferes with Golgi-dependent secretion inhibited the secretion of apoplastic effectors such as Bas4 and Slp1 but had no effect on the localization of cytoplasmic effectors Pwl2, Bas1, and Bas107 to the BIC . Furthermore, actin and microtubules essential for vesicle trafficking are required only for the secretion of apoplastic effectors but not for cytoplasmic effectors. Many genes involved in the ER to Golgi secretory pathway and post-translational modifications also are likely important for the secretion of apoplastic effectors in M. oryzae. For example, the LHS1 ER chaperone gene is required for the translocation and secretion of apoplastic effectors such as Slp1 .
Translocation of Effectors into Plant Cells
Several cytoplasmic effectors, including Pwl2, Bas1, and Avr-Piz-t, are translocated into the cytoplasm of rice cells , . Whereas some apoplastic effectors may be recognized by host plant surface receptors, the others that have intracellular targets, such as Avr1-CO39, also enter plant cells. In rice leaf sheath cells penetrated by fungal transformants expressing the Pwl2 and Avr-Piz-t proteins tagged with NLS and mRFP sequences, red fluorescence was observed in the nucleus of plant cells . However, the underlying molecular mechanism responsible for effector translocation into plant cells is not clear. In experiments with purified recombinant proteins, the native Avr1-CO39 protein was translocated into rice protoplasts, indicating that it can enter plant cells independent of fungal factors . In Oomycete pathogens such as Phytophthora species, the RXLR sequence is important for the translocation of effector proteins into plant cells . Nevertheless, there is no evidence for the presence of functional RXLR sequences in M. oryzae effector proteins.
Movement of Effectors in Plant Cells
In the rice leaf sheath cells penetrated by M. oryzae, the cytoplasmic effectors Pwl2 and Bas1, but not apoplastic effector Bas4, were moved to neighboring cells ahead of the invasive hyphae . Red fluorescence was observed in the nucleus of rice leaf sheath cells penetrated by a transformant expressing the PWL2-mCherry-NLS fusion construct and a number of surrounding cells without IH. Fungal effectors entering un-colonized plant cells may be able to suppress host defense responses or elicit susceptibility. In fact, it takes a shorter time (2 h) for IH to move through the subsequently invaded cells than in the first colonized cells (12 h) .
Interestingly, movement of cytoplasmic effectors from vein-associated cells into neighboring cells was rare and of low efficiency in comparison with effector movement among regular epidermal cells , suggesting that effector trafficking depends on rice cell types. In addition, unlike the 39.3 kD Pwl2-mCherry fusion, the 68.3 kD Pwl2-tdTomato protein was defective in the movement from the penetrated cells to surrounding cells . Therefore, cell-to-cell effector translocation also depends on the protein size. These results indicate that Magnaporthe effectors may be moved symplastically through plasmodesmata . The movement of effector proteins and invasion of neighboring cells by IH may be co-regulated because IH of M. oryzae preferentially contacted and crossed the plant cell wall at the pit fields .
Although more and more fungal effectors are being discovered, our understanding of effector delivery and cell-to-cell movement in planta is relatively limited in comparison with that of bacterial effectors. Studies in M. oryzae showed that some effectors may be secreted via an unconventional protein secretion system to the BIC . The BIC-like structures may also exist in other plant pathogenic fungi, particularly other hemibiotrophic pathogens. To date, no common plant entry sequence has been identified in fungal effectors, indicating that fungi may utilize a variety of mechanisms for effector translocation. M. oryzae and other fungal pathogens may have conserved mechanisms to recognize plasmodesmata for the movement of effector proteins and spreading of IH.
- 1. Kankanala P, Czymmek K, Valent B (2007) Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus. Plant Cell 19: 706–724.
- 2. Djamei A, Kahmann R (2012) Ustilago maydis: Dissecting the molecular interface between pathogen and plant. PLoS Pathog 8: e1002955
- 3. de Wit P, Mehrabi R, van den Burg HA, Stergiopoulos I (2009) Fungal effector proteins: past, present and future. Mol Plant Pathol 10: 735–747.
- 4. Bohnert HU, Fudal I, Dioh W, Tharreau D, Notteghem JL, et al. (2004) A putative polyketide synthase peptide synthetase from Magnaporthe grisea signals pathogen attack to resistant rice. Plant Cell 16: 2499–2513.
- 5. Park C, Chen S, Shirsekar G, Zhou B, Khang C, et al. (2012) The Magnaporthe oryzae effector Avrpiz-t targets the ring E3 ubiquitin ligase apip6 to suppress pathogen-associated molecular pattern-triggered immunity in rice. Plant Cell 24: 4748–4762.
- 6. Jia Y, McAdams SA, Bryan GT, Hershey HP, Valent B (2000) Direct interaction of resistance gene and avirulence gene products confers rice blast resistance. EMBO J 19: 4004–4014.
- 7. Khang CH, Park S-Y, Lee Y-H, Valent B, Kang S (2008) Genome organization and evolution of the AVR-Pita avirulence gene family in the Magnaporthe grisea species complex. Mol Plant Microbe Interact 21: 658–670.
- 8. Kang SC, Sweigard JA, Valent B (1995) The PWL host specificity gene family in the blast fungus Magnaporthe grisea. Mol Plant Microbe Interact 8: 939–948.
- 9. Peyyala R, Farman ML (2006) Magnaporthe oryzae isolates causing gray leaf spot of perennial ryegrass possess a functional copy of the AVR1-CO39 avirulence gene. Mol Plant Pathol 7: 157–165.
- 10. Yoshida K, Saitoh H, Fujisawa S, Kanzaki H, Matsumura H, et al. (2009) Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae. Plant Cell 21: 1573–1591.
- 11. Cesari S, Thilliez G, Ribot C, Chalvon V, Michel C, et al. (2013) The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors Avr-Pia and Avr1-CO39 by direct binding. Plant cell 25: 1463–1481.
- 12. Mentlak TA, Kombrink A, Shinya T, Ryder LS, Otomo I, et al. (2012) Effector-mediated suppression of chitin-triggered immunity by Magnaporthe oryzae is necessary for rice blast disease. Plant Cell 24: 322–335.
- 13. Mosquera G, Giraldo MC, Khang CH, Coughlan S, Valent B (2009) Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as biotrophy-associated secreted proteins in rice blast disease. Plant Cell 21: 1273–1290.
- 14. Saitoh H, Fujisawa S, Mitsuoka C, Ito A, Hirabuchi A, et al. (2012) Large-scale gene disruption in Magnaporthe oryzae identifies mc69, a secreted protein required for infection by monocot and dicot fungal pathogens. PLoS Pathog 8: e1002711
- 15. Chen S, Songkumarn P, Venu RC, Gowda M, Bellizzi M, et al. (2013) Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice. Mol Plant Microbe Interact 26: 191–202.
- 16. Giraldo MC, Dagdas YF, Gupta YK, Mentlak TA, Yi M, et al. (2013) Two distinct secretion systems facilitate tissue invasion by the rice blast fungus Magnaporthe oryzae. Nat Commun 4: 1996.
- 17. Khang CH, Berruyer R, Giraldo MC, Kankanala P, Park S-Y, et al. (2010) Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement. The Plant Cell Online 22: 1388–1403.
- 18. Mentlak TA, Talbot NJ, Kroj T (2011) Effector translocation and delivery by the rice blast fungus Magnaporthe oryzae. Effectors in Plant-Microbe Interactions 219–241.
- 19. Ribot C, Cesari S, Abidi I, Chalvon V, Bournaud C, et al. (2013) The Magnaporthe oryzae effector AVR1CO39 is translocated into rice cells independently of a fungal-derived machinery. Plant J 74: 1–12.
- 20. Jiang RH, Tripathy S, Govers F, Tyler BM (2008) RXLR effector reservoir in two Phytophthora species is dominated by a single rapidly evolving superfamily with more than 700 members. Proc Natl Acad Sci USA 105: 4874–4879.