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1. tRNA modification and codon usage control pathogen secretion in host cells

   https://doi.org/doi.org/10.21203/rs.3.rs-1690424/v1  

   Gang Li; Ziwen Gong; Nawaraj Dulal; Richard Wilson (June 2022, Research square)

   Animal and plant microbial pathogens deploy effector proteins and virulence factors to manipulate host cell innate immunity, often using unconventional secretion routes that are poorly understood. Transfer RNA (tRNA) anticodon modifications occur across taxa, but few biological functions are known. Here, in the devastating blast fungus Magnaporthe oryzae, we find that unconventional protein secretion in living host rice cells depends on tRNA modification and codon usage. Using gene deletions, mass spectrometry and live-cell imaging, 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. In M. oryzae, cytoplasmic effectors like Pwl2 and AVR-Pita are translocated into host cells via an unconventional secretion route; apoplastic effectors like Bas4 are secreted by the conventional ER-Golgi pathway. Loss of U34 thiolation abolished PWL2 and AVR-PITA (but not BAS4) mRNA translation in host cells. Paromomycin treatment, which increases near-cognate tRNA acceptance, restored Pwl2 and AVR-Pita production in U34 thiolation-deficient mutant strains. Synonymous AA- to ¬¬AG-ending codon changes remediated PWL2 mRNA translation in uba4; in UBA4+, expressing recoded PWL2 resulted in Pwl2 super-secretion that destabilized the microbe-host cell interface. Thus, wobble U34 tRNA thiolation and codon usage tune pathogen unconventional protein secretion in host cells. 

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2. An Allelic Variant of the Broad-Spectrum Blast Resistance Gene Ptr in Weedy Rice Is Associated with Resistance to the Most Virulent Blast Race IB-33

   https://doi.org/10.1094/PDIS-09-21-2043-RE  

   Zhao, Haijun; Liu, Yan; Jia, Melissa H.; Jia, Yulin (June 2022, Plant Disease)

   Rice resistance (R) genes have been effectively deployed to prevent blast disease caused by the fungal pathogen Magnaporthe oryzae, one of the most serious threats for stable rice production worldwide. Weedy rice competing with cultivated rice may carry novel or lost R genes. The quantitative trait locus qBR12.3b was previously mapped between two single nucleotide polymorphism markers at the 10,633,942-bp and 10,820,033-bp genomic positions in a black-hull-awned (BHA) weed strain using a weed-crop-mapping population under greenhouse conditions. In this study, we found a portion of the known resistance gene Ptr encoding a protein with four armadillo repeats and confers a broad spectrum of blast resistance. We then analyzed the sequences of the Ptr gene from weedy rice, Ptr^BHA, and identified a unique amino acid glutamine at protein position 874. Minor changes of protein conformation of the Ptr^BHAgene were predicted through structural analysis of Ptr^BHA, suggesting that the product of Ptr^BHAis involved in disease resistance. A gene-specific codominant marker HJ17-13 from Ptr^BHAwas then developed to distinguish alleles in weeds and crops. The Ptr^BHAgene existed in 207 individuals of the same mapping population, where qBR12.3b was mapped using this gene-specific marker. Disease reactions of 207 individuals and their parents to IB-33 were evaluated. The resistant individuals had Ptr^BHAwhereas the susceptible individuals did not, suggesting that HJ17-13 is reliable to predict qBR12.3b. Taken together, this newly developed marker, and weedy rice genotypes carrying qBR12.3b, are useful for blast improvement using marker assisted selection. 

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3. Terminating rice innate immunity induction requires a network of antagonistic and redox‐responsive E3 ubiquitin ligases targeting a fungal sirtuin

   https://doi.org/10.1111/nph.16365  

   Li, Gang; Qi, Xiaobo; Sun, Guangchao; Rocha, Raquel O.; Segal, Lauren M.; Downey, Katherine S.; Wright, Janet D.; Wilson, Richard A. (January 2020, New Phytologist)

   Summary Fungal phytopathogens can suppress plant immune mechanisms in order to colonize living host cells. Identifying all the molecular components involved is critical for elaborating a detailed systems‐level model of plant infection probing pathogen weaknesses; yet, the hierarchy of molecular events controlling fungal responses to the plant cell is not clear.Here we show how, in the blast fungusMagnaporthe oryzae, terminating rice innate immunity requires a dynamic network of redox‐responsive E3 ubiquitin ligases targeting fungal sirtuin 2 (Sir2), an antioxidation regulator required for suppressing the host oxidative burst.Immunoblotting, immunopurification, mass spectrometry and gene functional analyses showed that Sir2 levels responded to oxidative stress via a mechanism involving ubiquitination and three antagonistic E3 ubiquitin ligases: Grr1 and Ptr1 maintained basal Sir2 levels in the absence of oxidative stress; Upl3 facilitated Sir2 accumulation in response to oxidative stress. Grr1 and Upl3 interacted directly with Sir2 in a manner that decreased and scaled with oxidative stress, respectively.DeletingUPL3depleted Sir2 during growth in rice cells, triggering host immunity and preventing infection. OverexpressingSIR2in the Δupl3mutant remediated pathogenicity. Our work reveals how redox‐responsive E3 ubiquitin ligases inM. oryzaemediate Sir2 accumulation‐dependent antioxidation to modulate plant innate immunity and host susceptibility. 

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4. Magnaporthe oryzae nucleoside diphosphate kinase is required for metabolic homeostasis and redox‐mediated host innate immunity suppression

   https://doi.org/10.1111/mmi.14580  

   Rocha, Raquel O.; Wilson, Richard A. (September 2020, Molecular Microbiology)

   Abstract The fungusMagnaporthe oryzaecauses blast, the most devastating disease of cultivated rice. After penetrating the leaf cuticle,M. oryzaegrows as a biotroph in intimate contact with living rice epidermal cells before necrotic lesions develop. Biotrophic growth requires maintaining metabolic homeostasis while suppressing plant defenses, but the metabolic connections and requirements involved are largely unknown. Here, we characterized theM. oryzaenucleoside diphosphate kinase‐encoding geneNDK1and discovered it was essential for facilitating biotrophic growth by suppressing the host oxidative burst—the first line of plant defense. NDK enzymes reversibly transfer phosphate groups from tri‐ to diphosphate nucleosides. Correspondingly, intracellular nucleotide pools were perturbed inM. oryzaestrains lackingNDK1through targeted gene deletion, compared to WT. This affected metabolic homeostasis: TCA, purine and pyrimidine intermediates, and oxidized NADP⁺, accumulated in Δndk1. cAMP and glutathione were depleted. ROS accumulated in Δndk1hyphae. Functional appressoria developed on rice leaf sheath surfaces, but Δndk1invasive hyphal growth was restricted and redox homeostasis was perturbed, resulting in unsuppressed host oxidative bursts that triggered immunity. We conclude Ndk1 modulates intracellular nucleotide pools to maintain redox balance via metabolic homeostasis, thus quenching the host oxidative burst and suppressing rice innate immunity during biotrophy. 

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5. Evolution of Phytophthora infestans on its potato host since the Irish potato famine

   https://doi.org/10.1038/s41467-024-50749-4  

   Coomber, Allison; Saville, Amanda; Ristaino, Jean Beagle (December 2024, Nature Communications)

   Phytophthora infestans is a major oomycete plant pathogen, responsible for potato late blight, which led to the Irish Potato Famine from 1845–1852. Since then, potatoes resistant to this disease have been bred and deployed worldwide. Their resistance (R) genes recognize pathogen effectors responsible for virulence and then induce a plant response stopping disease progression. However, most deployed R genes are quickly overcome by the pathogen. We use targeted sequencing of effector and R genes on herbarium specimens to examine the joint evolution in both P. infestans and potato from 1845–1954. Currently relevant effectors are historically present in P. infestans, but with alternative alleles compared tomodern reference genomes. The historic FAM-1 lineage has the virulent Avr1 allele and the ability to break the R1 resistance gene before breeders deployed it in potato. The FAM-1 lineage is diploid, but later, triploid US-1 lineages appear. We show that pathogen virulence genes and host resistance genes have undergone significant changes since the Famine, from both natural and artificial selection. 

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