Disruption of the Interfacial Membrane Leads to Magnaporthe oryzae Effector Re-location and Lifestyle Switch During Rice Blast Disease

Abstract

To cause the devastating rice blast disease, the hemibiotrophic fungus Magnaporthe oryzae produces invasive hyphae (IH) that are enclosed in a plant-derived interfacial membrane, known as the extra-invasive hyphal membrane (EIHM), in living rice cells. Little is known about when the EIHM is disrupted and how the disruption contributes to blast disease. Here we show that the disruption of the EIHM correlates with the hyphal growth stage in first-invaded susceptible rice cells. Our approach utilized GFP that was secreted from IH as an EIHM integrity reporter. Secreted GFP (sec-GFP) accumulated in the EIHM compartment but appeared in the host cytoplasm when the integrity of the EIHM was compromised. Live-cell imaging coupled with sec-GFP and various fluorescent reporters revealed that the loss of EIHM integrity preceded shrinkage and eventual rupture of the rice vacuole. The vacuole rupture coincided with host cell death, which was limited to the invaded cell with presumed closure of plasmodesmata. We report that EIHM disruption and host cell death are landmarks that delineate three distinct infection phases (early biotrophic, late biotrophic, and transient necrotrophic phases) within the first-invaded cell before reestablishment of biotrophy in second-invaded cells. M. oryzae effectors exhibited infection phase-specific localizations, including entry of the apoplastic effector Bas4 into the host cytoplasm through the disrupted EIHM during the late biotrophic phase. Understanding how infection phase-specific cellular dynamics are regulated and linked to host susceptibility will offer potential targets that can be exploited to control blast disease.

Clathrin-mediated Endocytosis Facilitates Internalization ofMagnaporthe oryzaeEffectors into Rice Cells

“…Slowdown of the translocation system would appear consistent with the loss of activity in BICs as host cells fill with IH. Front-loading effector translocation from BICs would make sense because cell-to-cell movement of blast cytoplasmic effectors through plasmodesmata must occur in the early stages of host cell invasion while the EIHM enclosing the IH remains intact and plasmodesmata in the invaded cell remain open (Sakulkoo et al, 2018;Jones et al, 2021). Active BICs are surrounded by host cytoplasm with dynamic connections to the cell periphery (Khang et al, 2010) (Supplemental Movie S2), where cytoplasmic streaming would rapidly disperse effectors to pit fields containing plasmodesmata.…”

“…Specifically, the host vacuole remains intact, but shrinks as IH grow. Both the EIHM and vacuolar membrane become disrupted, indicating death of the invaded cell, around the time IH move into neighboring cells (Mochizuki et al, 2015;Jones et al, 2021). However, by this time, IH have established themselves in living neighboring cells.…”

“…Blast IH growing in living host cells are enclosed by extensions of the plant plasma membrane termed the extra-invasive hyphal membrane (EIHM). Colonized cells die as they fill with hyphae and the EIHM and host vacuolar membranes become disrupted, while the fungus moves on to colonize neighboring cells using the same strategy (Kankanala et al, 2007; Mochizuki et al, 2015; Sakulkoo et al, 2018; Jones et al, 2021). Each successful IH undergoeshyphal morphogenesis and is associated with differentiated interfacial zones after entering the host cell lumen (Kankanala et al, 2007; Khang et al, 2010).…”

“…In contrast, apoplastic effectors, such as the biotrophy-associated secreted protein 4 (Bas4), the LysM protein 1 (Slp1), and Bas113, are secreted via conventional golgi-mediated secretion, and they accumulate uniformly throughout the apoplastic compartment enclosed within the EIHM, outlining the entire IH without bleeding into the plant cell wall (Mosquera et al, 2009; Mentlak et al, 2012; Giraldo et al, 2013). Indeed, localization of secreted Bas4 fluorescent fusion proteins has become a reliable live cell imaging marker for the intactness of the EIHM around IH (Khang et al, 2010; Mochizuki et al, 2015; Jones et al, 2021). After filling the host cell, hyphal differentiation and BIC relocalization recur for each new hypha that enters a living neighbor cell (Khang et al, 2010; Sakulkoo et al, 2018).…”

Phytohormone Priming of Tomato Plants Evoke Differential Behavior in Rhizoctonia solani During Infection, With Salicylate Priming Imparting Greater Tolerance Than Jasmonate

“…Therefore, they are thwarted by a different set of defense responses activated by JA and ethylene signaling ( Glazebrook, 2005 ). The hemibiotrophic pathogens, on the other hand, deploy a coordinated strategy of infection where an initial biotrophic mode is followed by necrotrophic mode ( Hane et al, 2020 ; Précigout et al, 2020 ; Jones et al, 2021 ), separated by a short biotrophy-necrotrophy switch ( Chowdhury et al, 2017a ). Accordingly, the host also utilizes both SA and JA-mediated defense signaling pathways in a sophisticated and coordinated manner to counter these pathogens during different stages of infection ( Ding et al, 2011 ; Mencia et al, 2020 ).…”