A fungal core effector exploits the OsPUX8B.2-OsCDC48-6 module to suppress plant immunity.

Proteins containing a ubiquitin regulatory X (UBX) domain are cofactors of Cell Division Cycle 48 (CDC48) and function in protein quality control. However, whether and how UBX-containing proteins participate in host-microbe interactions remain unclear. Here we show that MoNLE1, an effector from the fungal pathogen Magnaporthe oryzae, is a core virulence factor that suppresses rice immunity by specifically interfering with OsPUX8B.2. The UBX domain of OsPUX8B.2 is required for its binding to OsATG8 and OsCDC48-6 and controls its 26 S proteasome-dependent stability. OsPUX8B.2 and OsCDC48-6 positively regulate plant immunity against blast fungus, while the high-temperature tolerance heat-shock protein OsBHT, a putative cytoplasmic substrate of OsPUX8B.2-OsCDC48-6, negatively regulates defense against blast infection. MoNLE1 promotes the nuclear migration and degradation of OsPUX8B.2 and disturbs its association with OsBHT. Given the high conservation of MoNLE1 among fungal isolates, plants with broad and durable blast resistance might be generated by engineering intracellular proteins resistant to MoNLE1.

Unconventional secretion of Magnaporthe oryzae effectors in rice cells is regulated by tRNA modification and codon usage control.

Microbial pathogens deploy effector proteins to manipulate host cell innate immunity, often using poorly understood unconventional secretion routes. Transfer RNA (tRNA) anticodon modifications are universal, but few biological functions are known. Here, in the rice blast fungus Magnaporthe oryzae, we show how unconventional effector secretion depends on tRNA modification and codon usage. We characterized the M. oryzae Uba4-Urm1 sulfur relay system mediating tRNA anticodon wobble uridine 2-thiolation (s2U34), a conserved modification required for efficient decoding of AA-ending cognate codons. Loss of s2U34 abolished the translation of AA-ending codon-rich messenger RNAs encoding unconventionally secreted cytoplasmic effectors, but mRNAs encoding endoplasmic reticulum-Golgi-secreted apoplastic effectors were unaffected. Increasing near-cognate tRNA acceptance, or synonymous AA- to AG-ending codon changes in PWL2, remediated cytoplasmic effector production in Δuba4. In UBA4+, expressing recoded PWL2 caused Pwl2 super-secretion that destabilized the host-fungus interface. Thus, U34 thiolation and codon usage tune pathogen unconventional effector secretion in host rice cells.

A protein kinase coordinates cycles of autophagy and glutaminolysis in invasive hyphae of the fungus Magnaporthe oryzae within rice cells.

The blast fungus Magnaporthe oryzae produces invasive hyphae in living rice cells during early infection, separated from the host cytoplasm by plant-derived interfacial membranes. However, the mechanisms underpinning this intracellular biotrophic growth phase are poorly understood. Here, we show that the M. oryzae serine/threonine protein kinase Rim15 promotes biotrophic growth by coordinating cycles of autophagy and glutaminolysis in invasive hyphae. Alongside inducing autophagy, Rim15 phosphorylates NAD-dependent glutamate dehydrogenase, resulting in increased levels of α-ketoglutarate that reactivate target-of-rapamycin (TOR) kinase signaling, which inhibits autophagy. Deleting RIM15 attenuates invasive hyphal growth and triggers plant immunity; exogenous addition of α-ketoglutarate prevents these effects, while glucose addition only suppresses host defenses. Our results indicate that Rim15-dependent cycles of autophagic flux liberate α-ketoglutarate - via glutaminolysis - to reactivate TOR signaling and fuel biotrophic growth while conserving glucose for antioxidation-mediated host innate immunity suppression.

Genome Sequence Resource of a Colletotrichum graminicola Field Strain from China.

The maize anthracnose stalk rot and leaf blight diseases caused by the fungal pathogen Colletotrichum graminicola is emerging as an important threat to corn production worldwide. In this work, we provide an improved genome assembly of a C. graminicola strain (TZ-3) by using the PacBio Sequel II and Illumina high-throughput sequencing technologies. The genome of TZ-3 consists of 36 contigs with a length of 59.3 Mb. After correction and evaluation with the Illumina sequencing data and BUSCO, this genome showed a high assembly quality and integrity. Gene annotation of this genome predicted 11,911 protein-coding genes, among which 983 secreted protein-coding genes and 332 effector genes were predicted. Compared with previous genomes of C. graminicola strains, TZ-3 genome is superior in nearly all parameters. The genome assembly and annotation will enhance our knowledge of the genetic makeup of the pathogen and molecular mechanisms underlying its pathogenicity and will provide valuable insights into genome variation across different regions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Two Magnaporthe appressoria-specific (MAS) proteins, MoMas3 and MoMas5, are required for suppressing host innate immunity and promoting biotrophic growth in rice cells.

In the devastating rice blast fungus Magnaporthe oryzae, six Magnaporthe appressoria-specific (MAS) proteins are encoded by MoGAS1, MoGAS2 and MoMAS3-MoMAS6. MoGAS1 and MoGAS2 were previously characterized as M. oryzae virulence factors; however, the roles of the other four genes are unknown. Here, we found that, although the loss of any MAS gene did not affect appressorial formation or vegetative growth, ∆Momas3 and ∆Momas5 mutant strains (but not the others) were reduced in virulence on susceptible CO-39 rice seedlings. Focusing on ∆Momas3 and ∆Momas5 mutant strains, we found that they could penetrate host leaf surfaces and fill the first infected rice cell but did not spread readily to neighbouring cells, suggesting they were impaired for biotrophic growth. Live-cell imaging of fluorescently labelled MoMas3 and MoMas5 proteins showed that during biotrophy, MoMas3 localized to the apoplastic compartment formed between fungal invasive hyphae and the plant-derived extra-invasive hyphal membrane while MoMas5 localized to the appressoria and the penetration peg. The loss of either MoMAS3 or MoMAS5 resulted in the accumulation of reactive oxygen species (ROS) in infected rice cells, resulting in the triggering of plant defences that inhibited mutant growth in planta. ∆Momas3 and ∆Momas5 biotrophic growth could be remediated by inhibiting host NADPH oxidases and suppressing ROS accumulation. Thus, MoMas3 and MoMas5 are novel virulence factors involved in suppressing host plant innate immunity to promote biotrophic growth.

Srk1 kinase, a SR protein-specific kinase, is important for sexual reproduction, plant infection and pre-mRNA processing in Fusarium graminearum.

SR protein-specific kinases (SRPKs) uniquely with a spacer region are important splicing factors from yeast to human. However, little is known about their biological functions in filamentous fungi. Therefore, we characterized a SRPK called SRK1 in wheat scab fungus Fusarium graminearum. Our data showed that Srk1 is required for vegetative growth, sexual reproduction and plant infection, and plays critical roles in pre-mRNA alternative splicing and gene expression. Remarkably, we found that Srk1 displayed dynamic shuttling between cytoplasm and the nucleus, which is regulated by the divergent spacer domain rather than its kinase activity, suggesting a regulatory mechanism for Srk1. Interestingly, Srk1-GFP also localized to the septal pores, indicating a possible role of Srk1 unrelated to mRNA processing. Although both K1 and K2 lobes of the kinase domain are essential for Srk1 functions, the K2 but not K1 lobe is responsible for the septal pore localization. Lastly, we established that Srk1 physically interacts with the two SR proteins, FgNpl3 and FgSrp1. Overall, our results indicated that SRK1 regulates fungal development, plant infection and mRNA processing by phosphorylation of other splicing factors including SR proteins, and the spacer domain regulates the functions of Srk1 by modulating its nucleocytoplasmic shuttling.