Calcium channel CNGC19 mediates basal defense signaling to regulate colonization by Piriformospora indica in Arabidopsis roots

Abstract

The activation of calcium signaling is a crucial event for perceiving environmental stress. Colonization by Piriformospora indica, a growth-promoting root endosymbiont, activates cytosolic Ca2+ in Arabidopsis roots. In this study, we examined the role and functional relevance of calcium channels responsible for Ca2+ fluxes. Expression profiling revealed that CYCLIC NUCLEOTIDE GATED CHANNEL 19 (CNGC19) is an early-activated gene, induced by unidentified components in P. indica cell-wall extract. Functional analysis showed that loss-of-function of CNGC19 resulted in growth inhibition by P.indica, due to increased colonization and loss of controlled fungal growth. The cngc19 mutant showed reduced elevation of cytosolic Ca2+ in response to P. indica cell-wall extract in comparison to the wild-type. Microbe-associated molecular pattern-triggered immunity was compromised in the cngc19 lines, as evidenced by unaltered callose deposition, reduced cis-(+)-12-oxo-phytodienoic acid, jasmonate, and jasmonoyl isoleucine levels, and down-regulation of jasmonate and other defense-related genes, which contributed to a shift towards a pathogenic response. Loss-of-function of CNGC19 resulted in an inability to modulate indole glucosinolate content during P. indica colonization. CNGC19-mediated basal immunity was dependent on the AtPep receptor, PEPR. CNGC19 was also crucial for P. indica-mediated suppression of AtPep-induced immunity. Our results thus demonstrate that Arabidopsis CNGC19 is an important Ca2+ channel that maintains a robust innate immunity and is crucial for growth-promotion signaling upon colonization by P. indica.

Piriformospora indica employs host’s putrescine for growth promotion in plants

“…1A). For tracking of colonization, a green fluorescent protein (GFP)-tagged P. indica strain was utilized (Hilbert et al, 2012, Jogawat et al, 2020), and we observed both the chlamydospores and fungal hyphae in root cortex at 40 dpi, indicating endophytic colonization (Fig. 1B).…”

“…S1). Simultaneously, tomato roots colonized with GFP-tagged P. indica (Hilbert et al, 2012, Jogawat et al, 2020) were also harvested for visualization of colonization under confocal microscope (Leica TCS M5). GFP-tagged P. indica were received from Prof. Ralf Oelmüller (Friedrich Schiller University, Jena, Germany).…”

“…For confocal microscopy, the tomato roots were colonized with GFP-tagged P.indica strain (Hilbert et al, 2012). At 40 dpi the roots were harvested and observed under a confocal microscope (Leica TCS M5) at an emission wavelength of 505-530 nm, 470 nm excitation and digital sectioning of 4-5 μm of root thickness (Jogawat et al, 2020). The relative amount of fungal DNA was quantified in 30 and 40 dpi roots using real time-qPCR utilizing UBI3 and P. indica Tef1 (Bütehorn et al, 2000).…”

“…The relative amount of fungal DNA was quantified in 30 and 40 dpi roots using real time-qPCR utilizing UBI3 and P. indica Tef1 (Bütehorn et al, 2000). Relative changes in fungal DNA content were calculated using C T of PiTef1 which were normalized by C T of UBI3 using ΔΔCT equation and P. indica DNA content in control roots (0 dpi) was defined as 1.0 (Vadassery et al, 2008, Jogawat et al, 2020). The primer pairs used in the gene expression studies are given in Table S1.…”

“…Induced systemic resistance by P.indica in host plants is mediated by jasmonic acid signaling, GA signaling and cytoplasmic function of NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1) (Stein et al, 2008, Cosme et al, 2016). Plants also regulate the colonization through activation of basal defense pathway via cyclic nucleotide gated channel (CNGC19), which ensures controlled P. indica colonization (Jacobs et al, 2011; Jogawat et al, 2020). Elevated levels of plant-secondary metabolite, indole glucosinolate also restrict the propagation of P.indica and balance its growth on plant roots (Lahrmann et al, 2015).…”