The bZIP transcription factor BIP1 of the rice blast fungus is essential for infection and regulates a specific set of appressorium genes.

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration.

The genetic identity of neighboring plants in intraspecific mixtures modulates disease susceptibility of both wheat and rice.

Mixing crop cultivars has long been considered as a way to control epidemics at the field level and is experiencing a revival of interest in agriculture. Yet, the ability of mixing to control pests is highly variable and often unpredictable in the field. Beyond classical diversity effects such as dispersal barrier generated by genotypic diversity, several understudied processes are involved. Among them is the recently discovered neighbor-modulated susceptibility (NMS), which depicts the phenomenon that susceptibility in a given plant is affected by the presence of another healthy neighboring plant. Despite the putative tremendous importance of NMS for crop science, its occurrence and quantitative contribution to modulating susceptibility in cultivated species remains unknown. Here, in both rice and wheat inoculated in greenhouse conditions with foliar fungal pathogens considered as major threats, using more than 200 pairs of intraspecific genotype mixtures, we experimentally demonstrate the occurrence of NMS in 11% of the mixtures grown in experimental conditions that precluded any epidemics. Thus, the susceptibility of these 2 major crops results from indirect effects originating from neighboring plants. Quite remarkably, the levels of susceptibility modulated by plant-plant interactions can reach those conferred by intrinsic basal immunity. These findings open new avenues to develop more sustainable agricultural practices by engineering less susceptible crop mixtures thanks to emergent but now predictable properties of mixtures.

Correction: The genetic identity of neighboring plants in intraspecific mixtures modulates disease susceptibility of both wheat and rice.

[This corrects the article DOI: 10.1371/journal.pbio.3002287.].

A major genetic locus in neighbours controls changes of gene expression and susceptibility in intraspecific rice mixtures.

Reports indicate that intraspecific neighbours alter the physiology of focal plants, and with a few exceptions, their molecular responses to neighbours are unknown. Recently, changes in susceptibility to pathogen resulting from such interactions were demonstrated, a phenomenon called neighbour-modulated susceptibility (NMS). However, the genetics of NMS and the associated molecular responses are largely unexplored. Here, we analysed in rice the modification of biomass and susceptibility to the blast fungus pathogen in the Kitaake focal genotype in the presence of 280 different neighbours. Using genome-wide association studies, we identified the loci in the neighbour that determine the response in Kitaake. Using a targeted transcriptomic approach, we characterized the molecular responses in focal plants co-cultivated with various neighbours inducing a reduction in susceptibility. Our study demonstrates that NMS is controlled by one major locus in the rice genome of its neighbour. Furthermore, we show that this locus can be associated with characteristic patterns of gene expression in focal plant. Finally, we propose an hypothesis where Pi could play a role in explaining this case of NMS. Our study sheds light on how plants affect the physiology in their neighbourhood and opens perspectives for understanding plant-plant interactions.

Evolution of the rice blast pathogen on spatially structured rice landraces maintains multiple generalist fungal lineages.

Traditional agrosystems, where humans, crops and microbes have coevolved over long periods, can serve as models to understand the ecoevolutionary determinants of disease dynamics and help the engineering of durably resistant agrosystems. Here, we investigated the genetic and phenotypic relationship between rice (Oryza sativa) landraces and their rice blast pathogen (Pyricularia oryzae) in the traditional Yuanyang terraces of flooded rice paddies in China, where rice landraces have been grown and bred over centuries without significant disease outbreaks. Analyses of genetic subdivision revealed that indica rice plants clustered according to landrace names. Three new diverse lineages of rice blast specific to the Yuanyang terraces coexisted with lineages previously detected at the worldwide scale. Population subdivision in the pathogen population did not mirror pattern of population subdivision in the host. Measuring the pathogenicity of rice blast isolates on landraces revealed generalist life history traits. Our results suggest that the implementation of disease control strategies based on the emergence or maintenance of a generalist lifestyle in pathogens may sustainably reduce the burden of disease in crops.

Growth-defence trade-off in rice: fast-growing and acquisitive genotypes have lower expression of genes involved in immunity.

Plant ecologists and molecular biologists have long considered the hypothesis of a trade-off between plant growth and defence separately. In particular, how genes thought to control the growth-defence trade-off at the molecular level relate to trait-based frameworks in functional ecology, such as the slow-fast plant economics spectrum, is unknown. We grew 49 phenotypically diverse rice genotypes in pots under optimal conditions and measured growth-related functional traits and the constitutive expression of 11 genes involved in plant defence. We also quantified the concentration of silicon (Si) in leaves to estimate silica-based defences. Rice genotypes were aligned along a slow-fast continuum, with slow-growing, late-flowering genotypes versus fast-growing, early-flowering genotypes. Leaf dry matter content and leaf Si concentrations were not aligned with this axis and negatively correlated with each other. Live-fast genotypes exhibited greater expression of OsNPR1, a regulator of the salicylic acid pathway that promotes plant defence while suppressing plant growth. These genotypes also exhibited greater expression of SPL7 and GH3.2, which are also involved in both stress resistance and growth. Our results do not support the hypothesis of a growth-defence trade-off when leaf Si and leaf dry matter content are considered, but they do when hormonal pathway genes are considered. We demonstrate the benefits of combining ecological and molecular approaches to elucidate the growth-defence trade-off, opening new avenues for plant breeding and crop science.

Jasmonic acid contributes to rice resistance against Magnaporthe oryzae.

BACKGROUND: The annual yield losses caused by the Rice Blast Fungus, Magnaporthe oryzae, range to the equivalent for feeding 60 million people. To ward off infection by this fungus, rice has evolved a generic basal immunity (so called compatible interaction), which acts in concert with strain-specific defence (so-called incompatible interaction). The plant-defence hormone jasmonic acid (JA) promotes the resistance to M. oryzae, but the underlying mechanisms remain elusive. To get more insight into this open question, we employ the JA-deficient mutants, cpm2 and hebiba, and dissect the JA-dependent defence signalling in rice for both, compatible and incompatible interactions. RESULTS: We observe that both JA-deficient mutants are more susceptible to M. oryzae as compared to their wild-type background, which holds true for both types of interactions as verified by cytological staining. Secondly, we observe that transcripts for JA biosynthesis (OsAOS2 and OsOPR7), JA signalling (OsJAZ8, OsJAZ9, OsJAZ11 and OsJAZ13), JA-dependent phytoalexin synthesis (OsNOMT), and JA-regulated defence-related genes, such as OsBBTI2 and OsPR1a, accumulate after fungal infection in a pattern that correlates with the amplitude of resistance. Thirdly, induction of defence transcripts is weaker during compatible interaction. CONCLUSION: The study demonstrates the pivotal role of JA in basal immunity of rice in the resistance to M. oryzae in both, compatible and incompatible interactions.

From cultivar mixtures to allelic mixtures: opposite effects of allelic richness between genotypes and genotype richness in wheat.

Agroecosystem diversification through increased crop genetic diversity could provide multiple services such as improved disease control or increased productivity. However, we still poorly understand how genetic diversity affects agronomic performance. We grew 179 inbred lines of durum wheat in pure stands and in 202 binary mixtures in field conditions. We then tested the effect of allelic richness between genotypes and genotype richness on grain yield and Septoria tritici blotch disease. Allelic richness was tested at 19K single nucleotide polymorphisms distributed along the durum wheat genome. Both genotype richness and allelic richness could be equal to 1 or 2. Mixtures were overall more productive and less diseased than their pure stand components. Yet, we identified one locus at which allelic richness between genotypes was associated with increased disease severity and decreased grain yield. The effect of allelic richness at this locus was stronger than the effect of genotype richness on grain yield (-7.6% vs +5.7%). Our results suggest that positive effects of crop diversity can be reversed by unfavourable allelic associations. This highlights the need to integrate genomic data into crop diversification strategies. More generally, investigating plant-plant interactions at the genomic level is promising to better understand biodiversity-ecosystem functioning relationships.

Genome-wide association of rice response to blast fungus identifies loci for robust resistance under high nitrogen.

BACKGROUND: Nitrogen fertilization is known to increase disease susceptibility, a phenomenon called Nitrogen-Induced Susceptibility (NIS). In rice, this phenomenon has been observed in infections with the blast fungus Magnaporthe oryzae. A previous classical genetic study revealed a locus (NIS1) that enhances susceptibility to rice blast under high nitrogen fertilization. In order to further address the underlying genetics of plasticity in susceptibility to rice blast after fertilization, we analyzed NIS under greenhouse-controlled conditions in a panel of 139 temperate japonica rice strains. A genome-wide association analysis was conducted to identify loci potentially involved in NIS by comparing susceptibility loci identified under high and low nitrogen conditions, an approach allowing for the identification of loci validated across different nitrogen environments. We also used a novel NIS Index to identify loci potentially contributing to plasticity in susceptibility under different nitrogen fertilization regimes. RESULTS: A global NIS effect was observed in the population, with the density of lesions increasing by 8%, on average, under high nitrogen fertilization. Three new QTL, other than NIS1, were identified. A rare allele of the RRobN1 locus on chromosome 6 provides robust resistance in high and low nitrogen environments. A frequent allele of the NIS2 locus, on chromosome 5, exacerbates blast susceptibility under the high nitrogen condition. Finally, an allele of NIS3, on chromosome 10, buffers the increase of susceptibility arising from nitrogen fertilization but increases global levels of susceptibility. This allele is almost fixed in temperate japonicas, as a probable consequence of genetic hitchhiking with a locus involved in cold stress adaptation. CONCLUSIONS: Our results extend to an entire rice subspecies the initial finding that nitrogen increases rice blast susceptibility. We demonstrate the usefulness of estimating plasticity for the identification of novel loci involved in the response of rice to the blast fungus under different nitrogen regimes.

Plant neighbour-modulated susceptibility to pathogens in intraspecific mixtures.

As part of a trend towards diversifying cultivated areas, varietal mixtures are subject to renewed interest as a means to manage diseases. Besides the epidemiological effects of varietal mixtures on pathogen propagation, little is known about the effect of intraspecific plant-plant interactions and their impact on responses to disease. In this study, genotypes of rice (Oryza sativa) or durum wheat (Triticum turgidum) were grown with different conspecific neighbours and manually inoculated under conditions preventing pathogen propagation. Disease susceptibility was measured together with the expression of basal immunity genes as part of the response to intra-specific neighbours. The results showed that in many cases for both rice and wheat susceptibility to pathogens and immunity was modified by the presence of intraspecific neighbours. This phenomenon, which we term 'neighbour-modulated susceptibility' (NMS), could be caused by the production of below-ground signals and does not require the neighbours to be infected. Our results suggest that the mechanisms responsible for reducing disease in varietal mixtures in the field need to be re-examined.

Heterogeneity of the rice microbial community of the Chinese centuries-old Honghe Hani rice terraces system.

The Honghe Hani rice terraces system (HHRTS) is a traditional rice cultivation system where Hani people cultivate remarkably diverse rice varieties. Recent introductions of modern rice varieties to the HHRTS have significantly increased the severity of rice diseases within the terraces. Here, we determine the impacts of these recent introductions on the composition of the rice-associated microbial communities. We confirm that the HHRTS contains a range of both traditional HHRTS landraces and introduced modern rice varieties and find differences between the microbial communities of these two groups. However, this introduction of modern rice varieties has not strongly impacted the overall diversity of the HHRTS rice microbial community. Furthermore, we find that the rice varieties (i.e. groups of closely related genotypes) have significantly structured the rice microbial community composition (accounting for 15%-22% of the variance) and that the core microbial community of HHRTS rice plants represents less than 3.3% of all the microbial taxa identified. Collectively, our study suggests a highly diverse HHRTS rice holobiont (host with its associated microbes) where the diversity of rice hosts mirrors the diversity of their microbial communities. Further studies will be needed to better determine how such changes might impact the sustainability of the HHRTS.

The genetics underlying natural variation of plant-plant interactions, a beloved but forgotten member of the family of biotic interactions.

Despite the importance of plant-plant interactions on crop yield and plant community dynamics, our understanding of the genetic and molecular bases underlying natural variation of plant-plant interactions is largely limited in comparison with other types of biotic interactions. By listing 63 quantitative trait loci (QTL) mapping and global gene expression studies based on plants directly challenged by other plants, we explored whether the genetic architecture and the function of the candidate genes underlying natural plant-plant interactions depend on the type of interactions between two plants (competition versus commensalism versus reciprocal helping versus asymmetry). The 16 transcriptomic studies are unevenly distributed between competitive interactions (n = 12) and asymmetric interactions (n = 4, all focusing on response to parasitic plants). By contrast, 17 and 30 QTL studies were identified for competitive interactions and asymmetric interactions (either weed suppressive ability or response to parasitic plants), respectively. Surprisingly, no studies have been carried out on the identification of genetic and molecular bases underlying natural variation in positive interactions. The candidate genes underlying natural plant-plant interactions can be classified into seven categories of plant function that have been identified in artificial environments simulating plant-plant interactions either frequently (photosynthesis, hormones), only recently (cell wall modification and degradation, defense pathways against pathogens) or rarely (ABC transporters, histone modification and meristem identity/life history traits). Finally, we introduce several avenues that need to be explored in the future to obtain a thorough understanding of the genetic and molecular bases underlying plant-plant interactions within the context of realistic community complexity.

Molecular Mechanisms Underlying Microbial Disease Control in Intercropping.

Many reports indicate that intercropping, which usually consists of growing two species next to each other, reduces the incidence of microbial diseases. Besides mechanisms operating at the field level, like inoculum dilution, there is recent evidence that plant-centered mechanisms with identified plant molecules and pathways are also involved. First, plants may trigger the induction of resistance in neighboring plants by the well-known mechanism of induced resistance. Second, molecules produced by one plant, either above- or belowground, can directly inhibit pathogens or indirectly trigger resistance through the induction of the plant immune system in neighboring plants. Third, competition for resources such as light or nutrients may indirectly modify the expression of the plant immune system. The conceptual frameworks of nonkin/stranger recognition and competition may be useful to further investigate the molecular mechanisms underlying crop protection in interspecific plant mixtures.

Cytokinin Production by the Rice Blast Fungus Is a Pivotal Requirement for Full Virulence.

Plants produce cytokinin (CK) hormones for controlling key developmental processes like source/sink distribution, cell division or programmed cell-death. Some plant pathogens have been shown to produce CKs but the function of this mimicry production by non-tumor inducing pathogens, has yet to be established. Here we identify a gene required for CK biosynthesis, CKS1, in the rice blast fungus Magnaporthe oryzae. The fungal-secreted CKs are likely perceived by the plant during infection since the transcriptional regulation of rice CK-responsive genes is altered in plants infected by the mutants in which CKS1 gene was deleted. Although cks1 mutants showed normal in vitro growth and development, they were severely affected for in planta growth and virulence. Moreover, we showed that the cks1 mutant triggered enhanced induction of plant defenses as manifested by an elevated oxidative burst and expression of defense-related markers. In addition, the contents of sugars and key amino acids for fungal growth were altered in and around the infection site by the cks1 mutant in a different manner than by the control strain. These results suggest that fungal-derived CKs are key effectors required for dampening host defenses and affecting sugar and amino acid distribution in and around the infection site.

Cuticular Defects in Oryza sativa ATP-binding Cassette Transporter G31 Mutant Plants Cause Dwarfism, Elevated Defense Responses and Pathogen Resistance.

The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between plant and environment. Homologs of the ATP-binding cassette (ABC) transporter AtABCG32/HvABCG31 clade are necessary for the formation of a functional cuticle in both monocots and dicots. Here we characterize the osabcg31 knockout mutant and hairpin RNA interference (RNAi)-down-regulated OsABCG31 plant lines having reduced plant growth and a permeable cuticle. The reduced content of cutin in leaves and structural alterations in the cuticle and at the cuticle-cell wall interface in plants compromised in OsABCG31 expression explain the cuticle permeability. Effects of modifications of the cuticle on plant-microbe interactions were evaluated. The cuticular alterations in OsABCG31-compromised plants did not cause deficiencies in germination of the spores or the formation of appressoria of Magnaporthe oryzae on the leaf surface, but a strong reduction of infection structures inside the plant. Genes involved in pathogen resistance were constitutively up-regulated in OsABCG31-compromised plants, thus being a possible cause of the resistance to M. oryzae and the dwarf growth phenotype. The findings show that in rice an abnormal cuticle formation may affect the signaling of plant growth and defense.

Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system.

Second-Site Mutagenesis of a Hypomorphic argonaute1 Allele Identifies SUPERKILLER3 as an Endogenous Suppressor of Transgene Posttranscriptional Gene Silencing.

Second-site mutagenesis was performed on the argonaute1-33 (ago1-33) hypomorphic mutant, which exhibits reduced sense transgene posttranscriptional gene silencing (S-PTGS). Mutations in FIERY1, a positive regulator of the cytoplasmic 5'-to-3' EXORIBONUCLEASE4 (XRN4), and in SUPERKILLER3 (SKI3), a member of the SKI complex that threads RNAs directly to the 3'-to-5' exoribonuclease of the cytoplasmic exosome, compensated AGO1 partial deficiency and restored S-PTGS with 100% efficiency. Moreover, xrn4 and ski3 single mutations provoked the entry of nonsilenced transgenes into S-PTGS and enhanced S-PTGS on partially silenced transgenes, indicating that cytoplasmic 5'-to-3' and 3'-to-5' RNA degradation generally counteract S-PTGS, likely by reducing the amount of transgene aberrant RNAs that are used by the S-PTGS pathway to build up small interfering RNAs that guide transgene RNA cleavage by AGO1. Constructs generating improperly terminated transgene messenger RNAs (mRNAs) were not more sensitive to ski3 or xrn4 than regular constructs, suggesting that improperly terminated transgene mRNAs not only are degraded from both the 3' end but also from the 5' end, likely after decapping. The facts that impairment of either 5'-to-3' or 3'-to-5' RNA degradation is sufficient to provoke the entry of transgene RNA into the S-PTGS pathway, whereas simultaneous impairment of both pathways is necessary to provoke the entry of endogenous mRNA into the S-PTGS pathway, suggest poor RNA quality upon the transcription of transgenes integrated at random genomic locations.

OsMADS26 Negatively Regulates Resistance to Pathogens and Drought Tolerance in Rice.

Functional analyses of MADS-box transcription factors in plants have unraveled their role in major developmental programs (e.g. flowering and floral organ identity) as well as stress-related developmental processes, such as abscission, fruit ripening, and senescence. Overexpression of the rice (Oryza sativa) MADS26 gene in rice has revealed a possible function related to stress response. Here, we show that OsMADS26-down-regulated plants exhibit enhanced resistance against two major rice pathogens: Magnaporthe oryzae and Xanthomonas oryzae. Despite this enhanced resistance to biotic stresses, OsMADS26-down-regulated plants also displayed enhanced tolerance to water deficit. These phenotypes were observed in both controlled and field conditions. Interestingly, alteration of OsMADS26 expression does not have a strong impact on plant development. Gene expression profiling revealed that a majority of genes misregulated in overexpresser and down-regulated OsMADS26 lines compared with control plants are associated to biotic or abiotic stress response. Altogether, our data indicate that OsMADS26 acts as an upstream regulator of stress-associated genes and thereby, a hub to modulate the response to various stresses in the rice plant.

The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding.

Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer-fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

The Magnaporthe oryzae effector AVR1-CO39 is translocated into rice cells independently of a fungal-derived machinery.

Effector proteins are key elements in plant-fungal interactions. The rice blast fungus Magnaporthe oryzae secretes numerous effectors that are suspected to be translocated inside plant cells. However, their cellular targets and the mechanisms of translocation are still unknown. Here, we have identified the open reading frame (ORF3) corresponding to the M. oryzae avirulence gene AVR1-CO39 that interacts with the rice resistance gene Pi-CO39 and encodes a small secreted protein without homology to other proteins. We demonstrate that AVR1-CO39 is specifically expressed and secreted at the plant-fungal interface during the biotrophic phase of infection. Live-cell imaging with M. oryzae transformants expressing a translational fusion between AVR1-CO39 and the monomeric red fluorescent protein (mRFP) indicated that AVR1-CO39 is translocated into the cytoplasm of infected rice cells. Transient expression of an AVR1-CO39 isoform without a signal peptide in rice protoplasts triggers a Pi-CO39-specific hypersensitive response, suggesting that recognition of AVR1-CO39 by the Pi-CO39 gene product occurs in the cytoplasm of rice cells. The native AVR1-CO39 protein enters the secretory pathway of rice protoplasts as demonstrated by the ER localization of AVR1-CO39:mRFP:HDEL translational fusions, and is correctly processed as shown by Western blotting. However, this secreted AVR1-CO39 isoform triggers a Pi-CO39-specific hypersensitive response and accumulates inside rice protoplasts as shown by Western blotting and localization of AVR1-CO39:mRFP translational fusions. This indicates that AVR1-CO39 is secreted by rice protoplasts and re-enters into the cytoplasm by unknown mechanisms, suggesting that translocation of AVR1-CO39 into rice cells occurs independently of fungal factors.