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

Abstract

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 fungus Magnaporthe 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. Deleting UPL3 depleted Sir2 during growth in rice cells, triggering host immunity and preventing infection. Overexpressing SIR2 in the Δupl3 mutant remediated pathogenicity. Our work reveals how redox‐responsive E3 ubiquitin ligases in M. oryzae mediate Sir2 accumulation‐dependent antioxidation to modulate plant innate immunity and host susceptibility.

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

“…To our knowledge, NDPKs have not been studied in phytopathogenic fungi with specialized infection strategies. Although a subsequent study did not confirm direct interactions between Sir2 and Ndk1 (Li et al ., 2020), we had by that time already generated a NDK1 knockout strain by homologous gene replacement in the Guy11 wild‐type (WT) isolate, and our preliminary results showed that when equal numbers of spores were applied to three‐week‐old seedlings of the susceptible rice cultivar CO‐39, the Δ ndk1 ‐carrying mutant strain was non‐pathogenic compared to WT and the Δ ndk1 NDK1 complementation strain (Figure 1a). Specifically, small, uniform dark brown pinpoint flecks without visible centers formed on leaves inoculated with all of our Δ ndk1 mutant strains, but they did not produce spores, and M. oryzae could not be isolated from them, indicating they were type I lesions arising from non‐pathogenic interactions (Valent et al ., 1991).…”

“…Otherwise, downstream plant immune defenses are triggered and infection is aborted. Many disparate cellular processes related to antioxidation or the mitigation of oxidative damage—for example the superoxide dismutase system, the glutathione antioxidation system, and the fungal nitrooxidative stress response (Fernandez and Wilson, 2014; Marroquin‐Guzman et al, 2017b; Li et al ., 2020)—maintain intracellular redox homeostasis in order to facilitate plant ROS neutralization. However, it is becoming increasingly clear that these processes act collectively to tightly regulate redox balance such that perturbing one component of this network collapses the entire ROS scavenging system, resulting in planta in an unchecked host oxidative burst.…”

“…A wide‐spread characteristic of many filamentous phytopathogens including fungi is their ability to grow (at least initially) in living host cells, surrounded by plant‐derived membranes. This occurs while secreting protein effectors and employing other tactics to prevent triggering plant innate immunity, such as neutralizing host reactive oxygen species (ROS) (Yi and Valent, 2013; Li et al ., 2020). For fungi, an unresolved problem lies in comprehensively understanding—at the molecular level—how invasive hyphal growth, metabolism, and sustained plant defense suppression are linked during this biotrophic stage of host colonization (Sun et al ., 2018; Li et al ., 2020).…”

“…NMO2 is involved in the nitrooxidative damage response (Marroquin‐Guzman et al ., 2017b). UPL3 encodes an E3 ubiquitin ligase controlling Sir2 accumulation (Li et al ., 2020). SIR2 is required (among other processes) for the expression of M. oryzae superoxide dismutase genes including SOD1 (Fernandez et al ., 2014).…”

“…This occurs while secreting protein effectors and employing other tactics to prevent triggering plant innate immunity, such as neutralizing host reactive oxygen species (ROS) (Yi and Valent, 2013; Li et al ., 2020). For fungi, an unresolved problem lies in comprehensively understanding—at the molecular level—how invasive hyphal growth, metabolism, and sustained plant defense suppression are linked during this biotrophic stage of host colonization (Sun et al ., 2018; Li et al ., 2020). Elaborating molecular connections between fungal biotrophic growth processes and plant susceptibility could point to novel mitigation strategies while informing basic principles of plant infection.…”