The syntaxin protein (MoSyn8) mediates intracellular trafficking to regulate conidiogenesis and pathogenicity of rice blast fungus.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate cellular membrane fusion and intracellular vesicle trafficking in eukaryotic cells, and are critical in the growth and development of pathogenic fungi such as Magnaporthe oryzae which causes rice blast. Rice blast is thought to involve distinct SNARE-mediated transport and secretion of fungal effector proteins into the host to modulate rice immunity. We have previously characterized two SNARE proteins, secretory protein (MoSec22) and vesicle-associated membrane protein (MoVam7), as being important in cellular transport and pathogenicity. Here, we show that syntaxin 8 (MoSyn8), a Qc-SNARE protein homolog, also plays important roles in growth, conidiation, and pathogenicity. The MoSYN8 deletion mutant (∆Mosyn8) mutant exhibits defects in endocytosis and F-actin organization, appressorium turgor pressure generation, and host penetration. In addition, the ∆Mosyn8 mutant cannot elaborate biotrophic invasion of the susceptible rice host, or secrete avirulence factors Avr-Pia (corresponding to the rice resistance gene Pia) and Avrpiz-t (the cognate Avr gene for the resistance gene Piz-t) proteins. Our study of MoSyn8 advances our understanding of SNARE proteins in effector secretion which underlies the normal physiology and pathogenicity of M. oryzae, and it sheds new light on the mechanism of the blight disease caused by M. oryzae.

Overexpression of a Phytophthora Cytoplasmic CRN Effector Confers Resistance to Disease, Salinity and Drought in Nicotiana benthamiana.

The Crinkler (CRN) effector family is produced by oomycete pathogens and may manipulate host physiological and biochemical events inside host cells. Here, PsCRN161 was identified from Phytophthora sojae based on its broad and strong cell death suppression activities. The effector protein contains two predicted nuclear localization signals and localized to nuclei of plant cells, indicating that it may target plant nuclei to modify host cell physiology and function. The chimeric gene GFP:PsCRN161 driven by the Cauliflower mosaic virus (CaMV) 35S promoter was introduced into Nicotiana benthamiana. The four independent PsCRN161-transgenic lines exhibited increased resistance to two oomycete pathogens (P. parasitica and P. capsici) and showed enhanced tolerance to salinity and drought stresses. Digital gene expression profiling analysis showed that defense-related genes, including ABC transporters, Cyt P450 and receptor-like kinases (RLKs), were significantly up-regulated in PsCRN161-transgenic plants compared with GFP (green fluorescent protein) lines, implying that PsCRN161 expression may protect plants from biotic and abiotic stresses by up-regulation of many defense-related genes. The results reveal previously unknown functions of the oomycete effectors, suggesting that the pathogen effectors could be directly used as functional genes for plant molecular breeding for enhancement of tolerance to biotic and abiotic stresses.

System-wide characterization of bZIP transcription factor proteins involved in infection-related morphogenesis of Magnaporthe oryzae.

The basic leucine zipper (bZIP) domain-containing transcription factors (TFs) function as key regulators of cellular growth and differentiation in eukaryotic organisms including fungi. We have previously identified MoAp1 and MoAtf1 as bZIP TFs in Magnaporthe oryzae and demonstrated that they regulate the oxidative stress response and are critical in conidiogenesis and pathogenicity. Studies of bZIP proteins could provide a novel strategy for controlling rice blast, but a systematic examination of the bZIP proteins has not been carried out. Here, we identified 19 additional bZIP TFs and characterized their functions. We found that the majority of these TFs exhibit active functions, most notably, in conidiogenesis. We showed that MoHac1 regulates the endoplasmic reticulum stress response through a conserved unfolded protein response pathway, MoMetR controls amino acid metabolism to govern growth and differentiation, and MoBzip10 governs appressorium function and invasive hyphal growth. Moreover, MoBzip5 participates in appressorium formation through a pathway distinct from that MoBzip10, and MoMeaB appears to exert a regulatory role through nutrient uptake and nitrogen utilization. Collectively, our results provide insights into shared and specific functions associated with each of these TFs and link the regulatory roles to the fungal growth, conidiation, appressorium formation, host penetration and pathogenicity.

MoLys2 is necessary for growth, conidiogenesis, lysine biosynthesis, and pathogenicity in Magnaporthe oryzae.

Amino acid biosyntheses are complex but essential processes in growth and differentiation of eukaryotic cells. In the budding yeast Saccharomyces cerevisiae, the lysine biosynthesis via the α-aminoadipate (AA) pathway involves several steps, including reduction of AA to AA 6-semialdehyde by AA reductase ScLys2. In filamentous fungus Penicillium chrysogenum, disruption of the LYS2 gene blocked the lysine biosynthesis but promoted the production of the secondary metabolite penicillin. In comparison, little is known about the function of AA reductase Lys2 in phytopathogenic fungi. We here characterized the functions of MoLys2, a homolog of ScLys2, from the rice blast fungus Magnaporthe oryzae. Our results showed that the ΔMolys2 mutants were auxotrophic for lysine. The ΔMolys2 mutants also exhibited drastic reduction in pathogenicity on rice, inducing small disease lesions. Microscopic examination of the lesions revealed that the invasive hyphae of ΔMolys2 mutants were mostly restricted to the primary infected leaf sheath cells. In addition, exogenous lysine restored the production of conidia and near wild-type appressoria differentiation, and rescued the defect of pathogenicity in conidia infection of detached barely and rice leaf sheath. Our results indicated that MoLys2 is necessary for lysine biosynthesis that affects growth, conidiogenesis, and pathogenicity of the fungus. This study does implicate the potential for targeting lysine biosynthesis for the development of novel fungicides against M. oryzae.

Two host cytoplasmic effectors are required for pathogenesis of Phytophthora sojae by suppression of host defenses.

Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler. Their functions and mechanisms in pathogenesis are mostly unknown. Here, we identify a group of five P. sojae-specific CRN-like genes with high levels of sequence similarity, of which three are putative pseudogenes. Functional analysis shows that the two functional genes encode proteins with predicted nuclear localization signals that induce contrasting responses when expressed in Nicotiana benthamiana and soybean (Glycine max). PsCRN63 induces cell death, while PsCRN115 suppresses cell death elicited by the P. sojae necrosis-inducing protein (PsojNIP) or PsCRN63. Expression of CRN fragments with deleted signal peptides and FLAK motifs demonstrates that the carboxyl-terminal portions of PsCRN63 or PsCRN115 are sufficient for their activities. However, the predicted nuclear localization signal is required for PsCRN63 to induce cell death but not for PsCRN115 to suppress cell death. Furthermore, silencing of the PsCRN63 and PsCRN115 genes in P. sojae stable transformants leads to a reduction of virulence on soybean. Intriguingly, the silenced transformants lose the ability to suppress host cell death and callose deposition on inoculated plants. These results suggest a role for CRN effectors in the suppression of host defense responses.

MoImd4 mediates crosstalk between MoPdeH-cAMP signalling and purine metabolism to govern growth and pathogenicity in Magnaporthe oryzae.

The high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance in the rice blast fungus Magnaporthe oryzae. To explore the underlying mechanism, we identified MoImd4 as an inosine-5'-monophosphate dehydrogenase (IMPDH) homologue that interacts with MoPdeH. Targeted deletion of MoIMD4 resulted in reduced de novo purine biosynthesis and growth, as well as attenuated pathogenicity, which were suppressed by exogenous xanthosine monophosphate (XMP). Treatment with mycophenolic acid (MPA), which specifically inhibits MoImd4 activity, resulted in reduced growth and virulence attenuation. Intriguingly, further analysis showed that MoImd4 promotes the phosphodiesterase activity of MoPdeH, thereby decreasing intracellular cAMP levels, and MoPdeH also promotes the IMPDH activity of MoImd4. Our studies revealed the presence of a novel crosstalk between cAMP regulation and purine biosynthesis in M. oryzae, and indicated that such a link is also important in the pathogenesis of M. oryzae.

Light-Induced Hydrogel Based on Tumor-Targeting Mesoporous Silica Nanoparticles as a Theranostic Platform for Sustained Cancer Treatment.

Herein, we report a facile fabrication of a polymer (azobenzene and α-cyclodextrin-functionalized hyaluronic acid) and gold nanobipyramids (AuNBs) conjugated mesoporous silica nanoparticles (MSNs) to be used as an injectable drug delivery system for sustained cancer treatment. Because of the specific affinity between the hyaluronic acid (HA) on MSNs and the CD44 antigen overexpressed on tumor cells, the MSNs can selectively attach to tumor cells. The nanocomposite material then exploits thermoresponsive interactions between α-cyclodextrin and azobenzene, and the photothermal properties of gold nanobipyramids, to in situ self-assemble into a hydrogel under near-infrared (NIR) radiation. Upon gelation, the drug (doxorubicin)-loaded MSNs carriers were enclosed in the HA network of the hydrogel, whereas further degradation of the HA in the hydrogel due to the upregulation of hyaluronidase (HAase) around the tumor tissue will result in the release of MSNs from the hydrogel, which can then be taken by tumor cells and deliver their drug to the cell nuclei. This design is able to provide a microenvironment with rich anticancer drugs in, and around, the tumor tissue for time periods long enough to prevent the recrudescence of the disease. The extra efficacy that this strategy affords builds upon the capabilities of conventional therapies.

Hyperbranched phosphoramidate-hyaluronan hybrid: a reduction-sensitive injectable hydrogel for controlled protein release.

In the study, an injectable hybrid hydrogel (HPPAE-HA) was fabricated by in situ polymerization between acrylated hyperbranched polyphosphoramidate (HPPAE-AC) and thiolated hyaluronan (HA-SH) via Michael-addition reaction. Gelation times ranged from 4 to 360 min, depending on the stoichiometric ratio of HA-SH to HPPAE-AC, HA concentration and solution pH. In addition, due to the readily oxidation of thiol groups, the HPPAE-HA hydrogel bears disulfide bond. The DTT triggered disulfide bond cleavage and the subsequent cross-linking between thiol and abundant acryloyl groups presented in HPPAE-HA hydrogel contribute to the controlled release of bovine serum albumin (BSA) from the hydrogels. The burst release of BSA from HPPAE-HA hydrogel decreased from 23.7 to 4.0% during the first 3 h, and the 80% cumulative release of BSA was retarded for 12 h to nearly 96 h in 10 mM DTT. The facile synthesis of Michael-additional, disulfide containing HPPAE-HA hydrogel may enable further development of hydrogel matrices potentially suitable for tissue engineering and drug delivery applications.

Phytophthora sojae effector PsCRN70 suppresses plant defenses in Nicotiana benthamiana.

Phytophthora sojae, an oomycete pathogen, produces a large number of effector proteins that enter into host cells. The Crinklers (Crinkling and Necrosis, CRN) are cytoplasmic effectors that are conserved in oomycete pathogens and their encoding genes are highly expressed at the infective stages in P. sojae. However, their roles in pathogenesis are largely unknown. Here, we functionally characterized an effector PsCRN70 by transiently and stably overexpressing it in Nicotiana benthamiana. We demonstrated that PsCRN70 was localized to the plant cell nucleus and suppressed cell death elicited by all the tested cell death-inducing proteins, including BAX, PsAvh241, PsCRN63, PsojNIP and R3a/Avr3a. Overexpression of the PsCRN70 gene in N. benthamiana enhanced susceptibility to P. parasitica. The H2O2 accumulation in the PsCRN70-transgenic plants was reduced compared to the GFP-lines. The transcriptional levels of the defense-associated genes, including PR1b, PR2b, ERF1 and LOX, were also down-regulated in the PsCRN70-transgenic lines. Our results suggest that PsCRN70 may function as a universal suppressor of the cell death induced by many elicitors, the host H2O2 accumulation and the expression of defense-associated genes, and therefore promotes pathogen infection.