Date Published: January 2, 2014
Publisher: Public Library of Science
Author(s): Shijie Zhang, Jin-Rong Xu, Joseph Heitman.
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 .
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 .
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 .
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 .
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.
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) .