Nicotiana benthamiana Calreticulin 3a Is Required for the Ethylene-Mediated Production of Phytoalexins and Disease Resistance Against Oomycete Pathogen Phytophthora infestans

Abstract

Mature Nicotiana benthamiana shows strong resistance to the potato late blight pathogen Phytophthora infestans. By screening using virus-induced random gene silencing, we isolated a gene for plant-specific calreticulin NbCRT3a as a required gene for resistance of N. benthamiana against P. infestans. NbCRT3a encodes an endoplasmic reticulum quality-control (ERQC) chaperone for the maturation of glycoproteins, including glycosylated cell-surface receptors. NbCRT3a-silenced plants showed no detectable growth defects but resistance to P. infestans was significantly compromised. Defense responses induced by the treatment with INF1 (a secretory protein of P. infestans), such as production of reactive oxygen species and accumulation of phytoalexins, were suppressed in NbCRT3a-silenced N. benthamiana. Expression of an ethylene-regulated gene for phytoalexin biosynthesis, NbEAS, was reduced in NbCRT3a-silenced plants, whereas the expression of salicylic acid-regulated NbPR-1a was not affected. Consistently, induction of ethylene production by INF1 was suppressed in NbCRT3a-silenced plants. Resistance reactions induced by a hyphal wall components elicitor prepared from P. infestans were also impaired in NbCRT3a-silenced plants. However, cell death induced by active mitogen-activated protein kinase kinase (NbMEK2(DD)) was not affected by the silencing of NbCRT3a. Thus, NbCRT3a is required for the initiation of resistance reactions of N. benthamiana in response to elicitor molecules derived from P. infestans.

The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana

“…Five silencing constructs (A1-190, A2-117, A2-158, A2-189, and A6-96) causing a significant reduction in resistance against P. infestans were found to encode full-size ABCG (PDR-type) transporters ( Figure 2B, Table 1). Further analyses of other lines not listed in Table 1 have been reported (Matsukawa et al, 2013;Ohtsu et al, 2014) or are in progress and will be reported elsewhere.…”

“…Given that N. benthamiana has an allopolyploid genome (Goodin et al, 2008), the two copies of Nb-ABCG1 were presumably derived from the Nicotiana species ancestral to N. benthamiana. In fact, we often find two copies of highly homologous genes in the genome sequence of N. benthamiana (Table 1; Matsukawa et al, 2013;Ohtsu et al, 2014). Because the nucleic acid sequences of the two Nb-ABCG1 genes were highly similar, we could not suppress expression of a single Nb-ABCG1 gene.…”

“…pollen coat layers but keep pollen viable (Doughty et al, 1993;Iwano et al, 2014) and capsidiol is soluble in cyclohexane (Matsukawa et al, 2013), we used cyclohexane to wash secreted capsidiol from INF1-treated leaves. Leakage of chlorophyll was not detected by washing with cyclohexane for 30 s, indicating that there was no major damage to leaf cells (Supplemental Figure 9).…”

“…Leaves of N. benthamiana were treated with purified capsidiol (1 mg/mL in 2% DMSO) (Matsukawa et al, 2013) by injecting the solution into the intracellular spaces of the abaxial sides of leaves using a needleless syringe.…”

“…In the solanaceaous model plant Nicotiana benthamiana (wild tobacco), the sesquiterpenoid phytoalexin, capsidiol, is produced from isopentenyl pyrophosphate (IPP) (McGarvey and Croteau, 1995;Matsukawa et al, 2013). IPP and its allylic isomer dimethylallyl pyrophosphate (DMAPP) are converted to farnesyl pyrophosphate (FPP), an intermediate in the biosynthesis of sterols and terpenoids (Figure 1).…”