UVI_02019870, a Puptive Effector from <i>Ustilaginoidea virens</i>, Interacts with a Chloroplastic-Like Protein OsCPL1

Ustilaginoidea virens, which causes rice false smut (RFS), is one of the most detrimental rice fungal diseases and poses a severe threat to rice production and quality. Effectors in U. virens often act as a group of essential virulence factors that play crucial roles in the interaction between host and the pathogen. Thus, the functions of individual effectors in U. virens need to be further explored. Here, we found a small secreted hypersensitive response-inducing protein UVI_02019870 was highly conserved in fungi. Furthermore, we performed Y2H and BiFC assay to demonstrated UVI_02019870 interacted with OsCPL1, which was predicted as a chloroplast precursor to regulate chloroplast signaling pathways. Our data provide a theory for gaining an insight into the molecular mechanisms underlying the UVI_02019870 virulence function.     

The core effector Cce1 is required for early infection of maize by Ustilago maydis

The biotrophic pathogen Ustilago maydis, the causative agent of corn smut disease, infects one of the most important crops worldwide – Zea mays. To successfully colonize its host, U. maydis secretes proteins, known as effectors, that suppress plant defense responses and facilitate the establishment of biotrophy. In this work, we describe the U. maydis effector protein Cce1. Cce1 is essential for virulence and is upregulated during infection. Through microscopic analysis and in vitro assays, we show that Cce1 is secreted from hyphae during filamentous growth of the fungus. Strikingly, Δcce1 mutants are blocked at early stages of infection and induce callose deposition as a plant defense response. Cce1 is highly conserved among smut fungi and the Ustilago bromivora ortholog complemented the virulence defect of the SG200Δcce1 deletion strain. These data indicate that Cce1 is a core effector with apoplastic localization that is essential for U. maydis to infect its host.     

Toward understanding of rice innate immunity against Magnaporthe oryzae

The blast fungus, Magnaporthe oryzae, causes serious disease on a wide variety of grasses including rice, wheat and barley. The recognition of pathogens is an amazing ability of plants including strategies for displacing virulence effectors through the adaption of both conserved and variable pathogen elicitors. The pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) were reported as two main innate immune responses in plants, where PTI gives basal resistance and ETI confers durable resistance. The PTI consists of extracellular surface receptors that are able to recognize PAMPs. PAMPs detect microbial features such as fungal chitin that complete a vital function during the organism’s life. In contrast, ETI is mediated by intracellular receptor molecules containing nucleotide-binding (NB) and leucine rich repeat (LRR) domains that specifically recognize effector proteins produced by the pathogen. To enhance crop resistance, understanding the host resistance mechanisms against pathogen infection strategies and having a deeper knowledge of innate immunity system are essential. This review summarizes the recent advances on the molecular mechanism of innate immunity systems of rice against M. oryzae. The discussion will be centered on the latest success reported in plant–pathogen interactions and integrated defense responses in rice.     

Functional modulation of an aquaporin to intensify photosynthesis and abrogate bacterial virulence in rice

Plant aquaporins are a recently noted biological resource with a great potential to improve crop growth and defense traits. Here, we report the functional modulation of the rice (Oryza sativa) aquaporin OsPIP1;3 to enhance rice photosynthesis and grain production and to control bacterial blight and leaf streak, the most devastating worldwide bacterial diseases in the crop. We characterize OsPIP1;3 as a physiologically relevant CO₂-transporting facilitator, which supports 30% of rice photosynthesis on average. This role is nullified by interaction of OsPIP1;3 with the bacterial protein Hpa1, an essential component of the Type III translocon that supports translocation of the bacterial Type III effectors PthXo1 and TALi into rice cells to induce leaf blight and streak, respectively. Hpa1 binding shifts OsPIP1;3 from CO₂ transport to effector translocation, aggravates bacterial virulence, and blocks rice photosynthesis. On the contrary, the external application of isolated Hpa1 to rice plants effectively prevents OsPIP1;3 from interaction with Hpa1 secreted by the bacteria that are infecting the plants. Blockage of the OsPIP1;3–Hpa1 interaction reverts OsPIP1;3 from effector translocation to CO₂ transport, abrogates bacterial virulence, and meanwhile induces defense responses in rice. These beneficial effects can combine to enhance photosynthesis by 29–30%, reduce bacterial disease by 58–75%, and increase grain yield by 11–34% in different rice varieties investigated in small-scale field trials conducted during the past years. Our results suggest that crop productivity and immunity can be coordinated by modulating the physiological and pathological functions of a single aquaporin to break the growth–defense tradeoff barrier.     

A nonclassically secreted effector of Magnaporthe oryzae targets host nuclei and plays important roles in fungal growth and plant infection

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases and poses a growing threat to food security worldwide. Like many other filamentous pathogens, rice blast fungus releases multiple types of effector proteins to facilitate fungal infection and modulate host defence responses. However, most of the characterized effectors contain an N-terminal signal peptide. Here, we report the results of the functional characterization of a nonclassically secreted nuclear targeting effector in M. oryzae (MoNte1). MoNte1 has no signal peptide, but can be secreted and translocated into plant nuclei driven by a nuclear targeting peptide. It could also induce hypersensitive cell death when transiently expressed in Nicotiana benthamiana. Deletion of the MoNTE1 gene caused a significant reduction of fungal growth and conidiogenesis, partially impaired appressorium formation and host colonization, and also dramatically attenuated the pathogenicity. Taken together, these findings reveal a novel effector secretion pathway and deepen our understanding of rice–M. oryzae interactions.     

Xanthomonas oryzae pv. oryzae XopQ protein suppresses rice immune responses through interaction with two 14-3-3 proteins but its phospho-null mutant induces rice immune responses and interacts with another 14-3-3 protein

Many bacterial phytopathogens employ effectors secreted through the type-III secretion system to suppress plant innate immune responses. The Xanthomonas type-III secreted non-TAL effector protein Xanthomonas outer protein Q (XopQ) exhibits homology to nucleoside hydrolases. Previous work indicated that mutations which affect the biochemical activity of XopQ fail to affect its ability to suppress rice innate immune responses, suggesting that the effector might be acting through some other pathway or mechanism. In this study, we show that XopQ interacts in yeast and in planta with two rice 14-3-3 proteins, Gf14f and Gf14g. A serine to alanine mutation (S65A) of a 14-3-3 interaction motif in XopQ abolishes the ability of XopQ to interact with the two 14-3-3 proteins and to suppress innate immunity. Surprisingly, the S65A mutant gains the ability to interact with a third 14-3-3 protein that is a negative regulator of innate immunity. The XopQS65A mutant is an inducer of rice immune responses and this property is dominant over the wild-type function of XopQ. Taken together, these results suggest that XopQ targets the rice 14-3-3 mediated immune response pathway and that its differential phosphorylation might enable interaction with alternative 14-3-3 proteins.     

PCR-based Specific Detection of Ustilaginoidea virens and Ephelis japonica

A PCR‐based technique for detection of clavicipitaceous pathogens in rice and related grasses was developed. The target pathogens were Ustilaginoidea virens, which causes rice false smut, and Ephelis japonica, which causes rice udbatta disease and black choke in grasses. To design specific primers, a comparison was made on genetic diversity on the rDNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene of U. virens, Ephelis japonica, as well as some other clavicipitaceous fungi. Each fungus was successfully detected by using a specific primer set with high sensitivity. Species‐specific primers designed here were capable of detecting these pathogens in plant tissues. The PCR detection was consistent with conventional histological observation. This nested PCR assay was sensitive and reliable for the detection of U. virens and E. japonica, and thus can be a used to study disease cycles and early prediction of false smut and udbatta‐disease incidence in fields.     

Identification and evaluation of potential bio-control fungal endophytes against <Emphasis Type="Italic">Ustilagonoidea virens</Emphasis> on rice plants

False smut disease of rice is posing an increasing concern for production, not only because of the hiking epidemic occurrence in rice production, but also because of the challenging specific pathogenesis of the disease. The aim of this work was to evaluate the potential of five fungal endophytes to reduce negative effects of rice false smut fungus (Ustilagonoidea virens) on rice plants, in both the laboratory and greenhouse. Though all the fungal isolates showed the ability to inhibit the growth of U. virens with varying degrees, isolate E337 showed significant antagonistic activity against the pathogenic fungi. The isolate E337 was identified as Antennariella placitae by molecular and morphological data analysis including 18S rDNA sequence analysis. This isolate showed a significant in vitro inhibition of mycelial growth of U. virens by dual culture method and it was subsequently tested for its in vivo biocontrol potential on false smut disease on rice plants. Greenhouse experiments confirmed that applications of conidia of A. placitae protected rice plants by improving rice yield and by decreasing the severity of false smut disease on susceptible rice plants. This is the first report where A. placitae has been identified as a biocontrol organism.     

Effector-mediated suppression of chitin-triggered immunity by magnaporthe oryzae is necessary for rice blast disease

Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue.     

The ‘pears and lemons’ protein UvPal1 regulates development and virulence of Ustilaginoidea virens

Ustilaginoidea virens is an economically important fungus causing a devastating grain disease, rice false smut. An insertional mutagenesis screen was used to explore biological mechanisms underlying infection process of U. virens. T184, a new mutant was identified, with abnormal conidial morphology and deficient virulence. Analysis of the T-DNA inserted gene UvPal1 in the mutant confirmed it as a putative homologue of a cellular morphogenetic protein in yeast, Pal1, whose function has not been well characterized. Deletion of UvPal1 affected hyphal growth, cell morphology, stress adaptation and virulence. UvPal1 could interact with the endocytic proteins, UvEde1 and UvSla2, but was not required for receptor-mediated endocytosis. A yeast two-hybrid (Y2H) analysis was further carried out to screen the UvPal1-interacting proteins, resulting in the identification of 16 putative interacting proteins. Interestingly, UvPal1 interacted with a septin protein, UvCdc11 in vivo and in vitro, and also affected subcellular localization of UvCdc11 protein. Deletion of the four core septins impaired the growth, morphogenesis, stress response and virulence. Collectively, effects on cell morphology, oxidative stress response and virulence are similar to those of UvPal1, suggesting that UvPal1 physically interacts with UvCdc11 to mediate the septin complex to maintain the cellular morphology and virulence of U. virens.     

Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.     

Identification and characterization of suppressors of plant cell death (SPD) effectors from Magnaporthe oryzae

Phytopathogenic microorganisms, including the fungal pathogen Magnaporthe oryzae, secrete a myriad of effector proteins to facilitate infection. Utilizing the transient expression of candidate effectors in the leaves of the model plant Nicotiana benthamiana, we identified 11 suppressors of plant cell death (SPD) effectors from M. oryzae that were able to block the host cell death reaction induced by Nep1. Ten of these 11 were also able to suppress BAX‐mediated plant cell death. Five of the 11 SPD genes have been identified previously as either essential for the pathogenicity of M. oryzae, secreted into the plant during disease development, or as suppressors or homologues of other characterized suppressors. In addition, of the remaining six, we showed that SPD8 (previously identified as BAS162) was localized to the rice cytoplasm in invaded and surrounding uninvaded cells during biotrophic invasion. Sequence analysis of the 11 SPD genes across 43 re‐sequenced M. oryzae genomes revealed that SPD2, SPD4 and SPD7 have nucleotide polymorphisms amongst the isolates. SPD4 exhibited the highest level of nucleotide diversity of any currently known effector from M. oryzae in addition to the presence/absence polymorphisms, suggesting that this gene is potentially undergoing selection to avoid recognition by the host. Taken together, we have identified a series of effectors, some of which were previously unknown or whose function was unknown, that probably act at different stages of the infection process and contribute to the virulence of M. oryzae.     

A bacterial type III effector targets plant vesicle-associated membrane proteins

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.     

Versatile effectors of phytopathogenic fungi target host immunity

Phytopathogenic fungi secrete a large arsenal of effector molecules, including proteinaceous effectors, small RNAs, phytohormones and derivatives thereof. The pathogenicity of fungal pathogens is primarily determined by these effectors that are secreted into host cells to undermine innate immunity, as well as to facilitate the acquisition of nutrients for their in planta growth and proliferation. After conventional and non-conventional secretion, fungal effectors are translocated into different subcellular compartments of the host cells to interfere with various biological processes. In extracellular spaces, apoplastic effectors cope with physical and chemical barriers to break the first line of plant defenses. Intracellular effectors target essential immune components on the plasma membrane, in the cytosol, including cytosolic organelles, and in the nucleus to suppress host immunity and reprogram host physiology, favoring pathogen colonization. In this review, we comprehensively summarize the recent advances in fungal effector biology, with a focus on the versatile virulence functions of fungal effectors in promoting pathogen infection and colonization. A perspective of future research on fungal effector biology is also discussed.