A Polygalacturonase-Inhibiting Protein from Grapevine Reduces the Symptoms of the Endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana Leaves Without Any Evidence for In Vitro Interaction

Abstract

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgipl gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.

Fungal Endopolygalacturonases Are Recognized as Microbe-Associated Molecular Patterns by the Arabidopsis Receptor-Like Protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1

“…We previously showed that PGs from B. cinerea are able to induce necrosis in tomato, broad bean, and N. benthamiana (Kars et al, 2005;Joubert et al, 2007). This response required enzyme activity, and there were marked differences in necrosisinducing activities between individual PGs, with BcPG2 causing massive and rapid tissue collapse (less than 30 min in broad bean) and BcPG3 causing no detectable symptoms in any of the three plants mentioned (Kars et al, 2005;Joubert et al, 2007). The response in Arabidopsis to PGs, therefore, appears to be fundamentally different from that in tomato, broad bean, and N. benthamiana.…”

“…For transient expression in Nicotiana benthamiana, Bcpg3 with a plant PR1 signal peptide was amplified from pAT2-3 (Joubert et al, 2007) with primers AT265/AT268 and cloned into donor vector pDONR207 to obtain the entry vector, which was checked by sequencing. Expression vectors were created by Gateway cloning into pSOL2095 or pGWB20 (Nakagawa et al, 2007) with the corresponding entry vectors and transformed into A. tumefaciens C58C1, carrying helper plasmid pCH32 , or into A. tumefaciens GV3101 (only for SERK2 and BAK1 constructs).…”

“…Deletion of either Bcpg1 or Bcpg2 resulted in a strong reduction in virulence on tomato and broad bean (Vicia faba) leaves (ten Have et al, 1998;Kars et al, 2005), presumably because the enzymes have a detrimental effect on the integrity of host cell walls and tissues. Indeed, infiltrating BcPG2 into broad bean leaves or transient expression of BcPG2 in Nicotiana benthamiana led to tissue collapse and necrosis, and the necrotic response was abolished when the catalytic domain of the PG was mutated (Kars et al, 2005;Joubert et al, 2007). By contrast, Poinssot et al (2003) reported that BcPG1 can activate plant defense responses in grapevine (Vitis vinifera) cell suspensions independently of its enzymatic activity, suggesting that the protein itself might be recognized by plant cells as an elicitor.…”

Integration of evolutionary and desolvation energy analysis identifies functional sites in a plant immunity protein

“…Indeed, while evolutionary analyses were made on PGIPs of different plant families, only one particular PGIP (PvPGIP2) was mutagenized and its inhibitory capability tested against a limited number of fungal PGs. Furthermore, because the in vitro assay conditions are unlikely to mimic the physiological microenvironment of the apoplast and the interaction between the variants and PGs might differ in planta from that detected in vitro (49), the effects of the variants obtained in this work on plant response against pathogens therefore cannot be easily predicted and remain to be determined.…”

Abscisic Acid Deficiency Causes Changes in Cuticle Permeability and Pectin Composition That Influence Tomato Resistance to Botrytis cinerea

“…To counteract the activity of fungal pectinases, many plants express polygalacturonase-inhibiting proteins and PME inhibitors, which are localized in the cell wall. The role of these proteins in plant defense against B. cinerea has been extensively demonstrated (Powell et al, 2000;Ferrari et al, 2003;Sicilia et al, 2005;Joubert et al, 2006Joubert et al, , 2007Lionetti et al, 2007). The interaction with the inhibitors not only limits the destructive potential of polygalacturonases but also leads to the accumulation of elicitoractive OGAs (De Lorenzo and Ferrari, 2002).…”