Structural Biology of Bacterial Type IV Secretion Systems.

Type IV secretion systems (T4SSs) are large multisubunit translocons, found in both gram-negative and gram-positive bacteria and in some archaea. These systems transport a diverse array of substrates from DNA and protein-DNA complexes to proteins, and play fundamental roles in both bacterial pathogenesis and bacterial adaptation to the cellular milieu in which bacteria live. This review describes the various biochemical and structural advances made toward understanding the biogenesis, architecture, and function of T4SSs.

Vacuolar recruitment of retromer by a SNARE complex enables infection-related trafficking in rice blast.

The retromer complex is a conserved sorting machinery that maintains cellular protein homeostasis by transporting vesicles containing cargo proteins to defined destinations. It is known to sort proteins at the vacuole membranes for retrograde trafficking, preventing their degradation in the vacuole. However, the detailed mechanism of retromer recruitment to the vacuole membrane has not yet been elucidated. Here, we show that the vacuolar SNARE complex MoPep12-MoVti1-MoVam7-MoYkt6 regulates retromer-mediated vesicle trafficking by recruiting the retromer to the vacuole membrane, which promotes host invasion in Magnaporthe oryzae. Such recruitment is also essential for the retrieval of the autophagy regulator MoAtg8 and enables appressorium-mediated host penetration. Furthermore, the vacuolar SNARE subunits are involved in suppressing the host defense response by regulating the deployment of retromer-MoSnc1-mediated effector secretion. Altogether, our results provide insights into the mechanism of vacuolar SNAREs-dependent retromer recruitment which is necessary for pathogenicity-related membrane trafficking events in the rice blast fungus.

Effector-triggered susceptibility by the rice blast fungus Magnaporthe oryzae.

Rice blast, the most destructive disease of cultivated rice world-wide, is caused by the filamentous fungus Magnaporthe oryzae. To cause disease in plants, M. oryzae secretes a diverse range of effector proteins to suppress plant defense responses, modulate cellular processes, and support pathogen growth. Some effectors can be secreted by appressoria even before host penetration, while others accumulate in the apoplast, or enter living plant cells where they target specific plant subcellular compartments. During plant infection, the blast fungus induces the formation of a specialized plant structure known as the biotrophic interfacial complex (BIC), which appears to be crucial for effector delivery into plant cells. Here, we review recent advances in the cell biology of M. oryzae-host interactions and show how new breakthroughs in disease control have stemmed from an increased understanding of effector proteins of M. oryzae are deployed and delivered into plant cells to enable pathogen invasion and host susceptibility.

Membrane component ergosterol builds a platform for promoting effector secretion and virulence in Magnaporthe oryzae.

The plasma membrane (PM) functions as a physical border between the extracellular and cytoplasmic environments that contribute to the interaction between host plants and pathogenic fungi. As a specific sterol constituent in the cell membrane, ergosterol plays a significant role in fungal development. However, the role of ergosterol in the infection of the rice blast fungus Magnaporthe oryzae remains unclear. In this study, we found that a sterol reductase, MoErg4, is involved in ergosterol biosynthesis and the regulation of plasma membrane integrity in M. oryzae. We found that defects in ergosterol biosynthesis disrupt lipid raft formation in the PM and cause an abnormal distribution of the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein MoSso1, inhibiting its interaction with the v-SNARE protein MoSnc1. In addition, we found that MoSso1-MoSnc1 interaction is important for biotrophic interface complex development and cytoplasmic effector protein secretion. Our findings suggested that ergosterol-enriched lipid rafts constitute a platform for interactions among various SNARE proteins that are required for the development and pathogenicity of M. oryzae.

Rab7/Retromer-based endolysosomal trafficking is essential for proper host invasion in rice blast.

Secretion is a fundamental process that plant pathogens utilize to deliver effectors into the host to downregulate immunity and promote infection. Here, we uncover a fascinating membrane trafficking and delivery route that originates from vacuolar membranes in Magnaporthe oryzae and conduits to the host interface and plasma membrane. To perform such secretory/trafficking function, MoRab7 first recruits the retromer complex to the vacuolar membrane, enabling recognition of a family of SNARE proteins, including MoSnc1. Live-cell imaging confirmed a highly dynamic vesicular trafficking of the retromer complex component(s) and MoSnc1 toward and across the host interface or plasma membrane, and subsequent fusion with target membranes. Interestingly, disruption of the MoRab7/Retromer/MoSnc1-based endolysosomal cascade affects effector secretion and fungal pathogenicity. Taken together, we discovered an unconventional protein and membrane trafficking route starting from the fungal endolysosomes to the M. oryzae-rice interaction interface and dissect the role of MoRab7/Retromer/MoSnc1 sorting machinery in effector secretion during biotrophy and invasive growth in rice blast fungus.

MoErv29 promotes apoplastic effector secretion contributing to virulence of the rice blast fungus Magnaporthe oryzae.

During plant-pathogenic fungi and host plants interactions, numerous pathogen-derived proteins are secreted resulting in the activation of the unfolded protein response (UPR) pathway. For efficient trafficking of secretory proteins, including those important in disease progression, the cytoplasmic coat protein complex II (COPII) exhibits a multifunctional role whose elucidation remains limited. Here, we discovered that the COPII cargo receptor MoErv29 functions as a target of MoHac1, a previously identified transcription factor of the UPR pathway. In Magnaporthe oryzae, deletion of MoERV29 severely affected the vegetative growth, conidiation and biotrophic invasion of the fungus in susceptible rice hosts. We demonstrated that MoErv29 is required for the delivery of secreted proteins through recognition and binding of the amino-terminal tripeptide motifs following the signal peptide. By using bioinformatics analysis, we predicted a cargo spectrum of MoErv29 and found that MoErv29 is required for the secretion of many proteins, including extracellular laccases and apoplastic effectors. This secretion is mediated through the conventional endoplasmic reticulum-Golgi secretion pathway and is important for conferring host recognition and disease resistance. Taken together, our results revealed how MoErv29 operates on effector secretion, and our findings provided a critical link between COPII vesicle trafficking and the UPR pathway.

A conserved co-chaperone is required for virulence in fungal plant pathogens.

The maize pathogenic fungus Ustilago maydis experiences endoplasmic reticulum (ER) stress during plant colonization and relies on the unfolded protein response (UPR) to cope with this stress. We identified the U. maydis co-chaperone, designated Dnj1, as part of this conserved cellular response to ER stress. ∆dnj1 cells are sensitive to the ER stressor tunicamycin and display a severe virulence defect in maize infection assays. A dnj1 mutant allele unable to stimulate the ATPase activity of chaperones phenocopies the null allele. A Dnj1-mCherry fusion protein localizes in the ER and interacts with the luminal chaperone Bip1. The Fusarium oxysporum Dnj1 ortholog contributes to the virulence of this fungal pathogen in tomato plants. Unlike the human ortholog, F. oxysporum Dnj1 partially rescues the virulence defect of the Ustilago dnj1 mutant. By enabling the fungus to restore ER homeostasis and maintain a high secretory activity, Dnj1 contributes to the establishment of a compatible interaction with the host. Dnj1 orthologs are present in many filamentous fungi, but are absent in budding and fission yeasts. We postulate a conserved and essential role during virulence for this class of co-chaperones.

The Type III Accessory Protein HrpE of Xanthomonas oryzae pv. oryzae Surpasses the Secretion Role, and Enhances Plant Resistance and Photosynthesis.

Many species of plant-pathogenic gram-negative bacteria deploy the type III (T3) secretion system to secrete virulence components, which are mostly characteristic of protein effectors targeting the cytosol of the plant cell following secretion. Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen causing bacterial blight disease, uses the T3 accessory protein HrpE to assemble the pilus pathway, which in turn secretes transcription activator-like (TAL) effectors. The hrpE gene can execute extensive physiological and pathological functions beyond effector secretion. As evidenced in this study, when the hrpE gene was deleted from the Xoo genome, the bacteria incur seriouimpairments in multiplication, motility, and virulence. The virulence nullification is attributed to reduced secretion and translocation of PthXo1, which is a TAL effector that determines the bacterial virulence in the susceptible rice varieties. When the HrpE protein produced by prokaryotic expression is applied to plants, the recombinant protein is highly effective at inducing the defense response. Moreover, leaf photosynthesis efficiency is enhanced in HrpE-treated plants. These results provide experimental avenues to modulate the plant defense and growth tradeoff by manipulating a bacterial T3 accessory protein.

The Small GTPase MoSec4 Is Involved in Vegetative Development and Pathogenicity by Regulating the Extracellular Protein Secretion in Magnaporthe oryzae.

The Rab GTPase proteins play important roles in the membrane trafficking, and consequently protein secretion and development of eukaryotic organisms. However, little is known about the function of Rab GTPases in Magnaporthe oryzae. To further explore the function of Rab GTPases, we deleted the ortholog of the yeast Sec4p protein in M. oryzae, namely MoSEC4. The ΔMosec4 mutant is defective in polarized growth and conidiation, and it displays decreased appressorium turgor pressure and attenuated pathogenicity. Notably, the biotrophic invasive hyphae produced in rice cells are more bulbous and compressed in the ΔMosec4 mutant. Further studies showed that deletion of the MoSEC4 gene resulted in decreased secretion of extracellular enzymes and mislocalization of the cytoplasmic effector PWL2-mCherry-NLS. In accordance with a role in secretion, the GFP-MoSec4 fusion protein mainly accumulates at tips of growing vegetative hyphae. Our results suggest that the MoSec4 protein plays important roles in the secretion of extracellular proteins and consequently hyphal development and pathogenicity in the rice blast fungus.

Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris.

It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris

It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris

It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.