Candidate Effector Proteins from the Maize Tar Spot Pathogen Phyllachora maydis Localize to Diverse Plant Cell Compartments

Abstract

Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmECs) proteins remain unknown and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super Yellow Fluorescent Protein (sYFP)-tagged P. maydis effector candidate proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane while an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest effector candidate proteins from P. maydis target diverse cellular organelles and may thus provide valuable insights into their putative functions as well as host processes potentially manipulated by this fungal pathogen.

Genome-informed trophic classification and functional characterization of virulence proteins from the maize tar spot pathogenPhyllachora maydis

“…Candidate effectors were selected by mining an improved genome assembly of P. maydis (assembly ASM2933922v1) (isolate PM02)and accompanying transcriptome (BioProject PRJNA928553) for proteins that encoded effector-like sequence characteristics, and which are expressed during primary disease formation (Helm et al, 2022; MacCready et al, 2023). Overall, we selected the top eighteen genes that fulfilled our selection criteria (Table 2; Supplemental Table 1).…”

“…Agroinfiltrated N. benthamiana leaf tissue was collected, and flash frozen in liquid nitrogen twenty-four and forty-eight hours post-agroinfiltration, and total protein was extracted as previously described by Helm et al, (2022)…”

“…Attenuation of ROS production in N. benthamiana was performed as previously described using a luminol-based chemiluminescence assay and chitin as an elicitor (Jaiswal et al, 2022). N. benthamiana leaf discs (5mm diameter) transiently expressing either free sYFP (3xHA:sYFP; Helm et al, 2022) or the candidate effector-fluorescent protein fusions were sampled two days post-agroinfiltration using a cork borer, washed Rogers and Jaiswal et al 14 three times in deionized water, and incubated overnight in sterile water in a 96-well OptiPlate TM microplate (Perkin Elmer). The following day, the deionized water was replaced with chitin elicitation solution (luminol [30μg/mL], horseradish peroxidase [20μg/mL], chitin [hexamer] [5μg/mL], and nuclease-free water) and ROS production was monitored by chemiluminescence for 40 minutes in a microplate reader (Tecan Infinite M200 Pro), with higher luminescence values indicating greater ROS production.…”

“…Moreover, elucidating the trophic lifestyle as well as the molecular mechanisms that contribute to P. maydis pathogenicity remains challenging as its reported obligate lifestyle hinders genetic manipulation (Helm et al, 2022; Rocco da Silva et al, 2021; Telenko et al, 2020). MacCready et al, (2023) recently reported a high-quality P. maydis isolate PM02 genome (assembly ASM2933922v1) and accompanying transcriptome from primary tar spot lesions during early disease development.…”

Small secreted effector protein from Fusarium sacchari suppresses host immune response by inhibiting ScPi21‐induced cell death

“…tritici (Wang et al, 2021); Verticillium dahliae ; Phyllachora maydis (Helm et al, 2022); Fusarium graminearum (Yang et al, 2021); Fusarium oxysporum f. sp. lycopersici (Houterman et al, 2009); Phytophthora pathogens (Haas et al, 2009;Jing et al, 2016;Lee et al, 2014).…”