The Bamboo mosaic virus (BaMV) is a positive-sense, single-stranded RNA virus. Previously, we identified that the chloroplast phosphoglycerate kinase (chl-PGK) from Nicotiana benthamiana is one of the viral RNA binding proteins involved in the BaMV infection cycle. Because chl-PGK is transported to the chloroplast, we hypothesized that chl-PGK might be involved in viral RNA localization in the chloroplasts. To test this hypothesis, we constructed two green fluorescent protein (GFP)-fused mislocalized PGK mutants, the transit peptide deletion mutant (NO TRANSIT PEPTIDE [NOTP]-PGK-GFP) and the nucleus location mutant (nuclear location signal [NLS]-PGK-GFP). Using confocal microscopy, we demonstrated that NOTP-PGK-GFP and NLS-PGK-GFP are localized in the cytoplasm and nucleus, respectively, in N. benthamiana plants. When NOTP-PGK-GFP and NLS-PGK-GFP are transiently expressed, we observed a reduction in BaMV coat protein accumulation to 47% and 27% that of the wild-type PGK-GFP, respectively. To localize viral RNA in infected cells, we employed the interaction of NLS-GFP-MS2 (phage MS2 coat protein) with the modified BaMV RNA containing the MS2 coat protein binding sequence. Using confocal microscopy, we observed that BaMV viral RNA localizes to chloroplasts. Furthermore, elongation factor1a fused with the transit peptide derived from chl-PGK or with a Rubisco small subunit can partially restore BaMV accumulation in NbPGK1-knockdown plants by helping BaMV target chloroplasts.

Summary: A lternaria species produce and excrete dimethyl coprogen siderophores to acquire iron. The A lternaria alternata gene AaNPS6 , encoding a polypeptide analogous to fungal nonribosomal peptide synthetases, was found to be required for the production of siderophores and virulence on citrus. Siderophores purified from culture filtrates of the wild‐type strain did not induce any phytotoxicity on the leaves of citrus. Fungal strains lacking AaNPS6 produced little or no detectable extracellular siderophores and displayed an increased sensitivity to H ₂ O ₂ , superoxide‐generating compounds ( KO ₂ and menadione) and iron depletion. Δ nps6 mutants were also defective for the production of melanin and conidia. The introduction of a wild‐type AaNPS6 under the control of its endogenous promoter to a Δ nps6 null mutant at least partially restored siderophore production and virulence to citrus, demonstrating a functional link between iron uptake and fungal pathogenesis. Elevated sensitivity to H ₂ O ₂ , seen for the Δ nps6 null strain could be relieved by exogenous application of ferric iron. The expression of the AaNPS6 gene was highly up‐regulated under low‐iron conditions and apparently controlled by the redox‐responsive yeast transcriptional regulator YAP1 . Hence, the maintenance of iron homeostasis via siderophore‐mediated iron uptake also plays an important role in resistance to toxic reactive oxygen species ( ROS ). Our results demonstrate further the critical role of ROS detoxification for the pathogenicity of A . alternata in citrus.

Major Facilitator Superfamily (MFS) transporters play an important role in multidrug resistance in fungi. We report an AaMFS19 gene encoding a MFS transporter required for cellular resistance to oxidative stress and fungicides in the phytopathogenic fungus Alternaria alternata. AaMFS19, containing 12 transmembrane domains, displays activity toward a broad range of substrates. Fungal mutants lacking AaMFS19 display profound hypersensitivities to cumyl hydroperoxide, potassium superoxide, many singlet oxygen-generating compounds (eosin Y, rose Bengal, hematoporphyrin, methylene blue, and cercosporin), and the cell wall biosynthesis inhibitor, Congo red. AaMFS19 mutants also increase sensitivity to copper ions, clotrimazole, fludioxonil, and kocide fungicides, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA). AaMFS19 mutants induce smaller necrotic lesions on leaves of a susceptible citrus cultivar. All observed phenotypes in the mutant are restored by introducing and expressing a wild-type copy of AaMFS19. The wild-type strain of A. alternata treated with either CHP or TIBA reduces radial growth and formation and germination of conidia, increases hyphal branching, and results in decreased expression of the AaMFS19 gene. The expression of AaMFS19 is regulated by the Yap1 transcription activator, the Hog1 and Fus3 mitogen-activated protein (MAP) kinases, the ‘two component’ histidine kinase, and the Skn7 response regulator. Our results demonstrate that A. alternata confers resistance to different chemicals via a membrane-bound MFS transporter.

Alternaria alternata relies on the ability to produce a host-selective toxin and to detoxify reactive oxygen species to successfully colonize the host. An A. alternata major facilitator superfamily transporter designated AaMFS54 was functionally characterized by analysis of loss- and gain-of-function mutations to better understand the factors required for fungal pathogenesis. AaMFS54 was originally identified from a wild-type expression library after being subtracted with that of a mutant impaired for the oxidative stress-responsive transcription regulator Yap1. AaMFS54 contains 14 transmembrane helixes. Fungal mutant lacking AaMFS54 produced fewer conidia and increased sensitivity to many potent oxidants (potassium superoxide and singlet-oxygen generating compounds), xenobiotics (2,3,5-triiodobenzoic acid and 2-chloro-5-hydroxypyridine), and fungicides (clotrimazole, fludioxonil, vinclozolin, and iprodione). AaMFS54 mutant induced necrotic lesion sizes similar to those induced by wild-type on leaves of susceptible citrus cultivars after point inoculation with spore suspensions. However, the mutant produced smaller colonies and less fluffy hyphae on the affected leaves. Virulence assays on citrus leaves inoculated by spraying with spores revealed that AaMFS54 mutant induced less severe lesions than wild-type, indicating the requirement of AaMFS54 in pathogenesis. All defective phenotypes were restored in a strain re-acquiring a functional copy of AaMFS54. Northern blotting analysis revealed that the expression of AaMFS54 was suppressed by xenobiotics. The current studies indicate that the Yap1-mediated transporter plays a role in resistance to toxic oxidants and fungicides in A. alternata. The relationships of MFS transporters with other regulatory components conferring stress resistance and A. alternata pathogenesis are also discussed.

The ability of the necrotrophic fungus Alternaria alternata to detoxify reactive oxygen species (ROS) is crucial for pathogenesis to citrus. We report regulation of siderophore-mediated iron acquisition and ROS resistance by the NADPH oxidase (NOX), the redox activating yes-associated protein 1 (YAP1) regulator, and the high-osmolarity glycerol 1 (HOG1) mitogen-activated protein kinase (MAPK). The A. alternata nonribosomal peptide synthetase (NPS6) is essential for the biosynthesis of siderophores, contributing to iron uptake under low-iron conditions. Fungal strains impaired for NOX, YAP1, HOG1 or NPS6 all display increased sensitivity to ROS. Exogenous addition of iron at least partially rescues ROS sensitivity seen for NPS6, YAP1, HOG1, and NOX mutants. Importantly, expression of the NPS6 gene and biosynthesis of siderophores are regulated by NOX, YAP1 and HOG1, supporting a functional link among these regulatory pathways. Although iron fully rescues H₂O₂ sensitivity seen in mutants impaired for the response regulator SKN7, neither expression of NPS6 nor biosynthesis of siderophores is controlled by SKN7. Our results indicate that the acquisition of environmental iron has profound effects on ROS detoxification.