Role of a xyloglucan-specific endo-beta-1,4-glucanase inhibitor in the interactions of Nicotiana benthamiana with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci

Abstract

A xyloglucan-specific endo-beta-1,4-glucanase inhibitor cDNA, NbXEGIP1, was amplified from diseased leaves of Nicotiana benthamiana. The sequence was similar to the tomato xyloglucan-specific endo-beta-1,4-glucanase inhibitor (XEGIP) and tobacco nectarin IV genes that have been described as binding and inactivating fungal Family 12 xyloglucan-specific endo-beta-1,4-glucanases. Expression of NbXEGIP1 was not detected in healthy leaves, but the gene was induced during the later stages of infection by the fungi Colletotrichum destructivum and C. orbiculare. Induction of NbXEGIP1 also occurred during disease development by the bacterium Pseudomonas syringae pv. tabaci and during the hypersensitive response produced by P. syringae pv. tabaci expressing avrPto. A portion of NbXEGIP1 was cloned into a tobacco rattle virus vector for virus-induced gene silencing in N. benthamiana. Silencing NbXEGIP1 did not affect the interactions with either Colletotrichum species but did significantly reduce population levels of P. syringae pv. tabaci in the compatible interaction and P. syringae pv. tabaci expressing avrPto in the incompatible interaction. In the susceptible response to P. syringae pv. tabaci, silencing of NbXEGIP1 also resulted in visibly wilted leaves several hours prior to necrosis, which was not observed in control plants. This was related to a significantly higher level of electrolyte leakage and higher expression of a defensin gene from infected NbXEGIP1-silenced leaves compared with control leaves. Silencing appeared to be specific as it did not affect expression of a related gene, NbXEGIP2. NbXEGIP1 may act as an inhibitor of a bacterial enzyme that degrades the xyloglucan-cellulose plant cell-wall network, and degradation of the cell wall results in host membrane disruption and signalling of defence responses.

Figure 1

Comparison of xyloglucanase inhibitor protein sequences from Solanaceae as well as the carrot EDGP protein. Wheat TAXI‐IV (BAD72882) was included as an outgroup. Amino acid sequences were aligned using CLUSTALX and a tree generated with the bootstrap neighbour‐joining‐tree procedure of CLUSTALX. Boostrap values represent the number of times out of 1000 that the branch was supported by bootstrap analysis. A description of each sequence is provided in Table 1.

Figure 2

Relative RT‐PCR of NbXEGIP1 expression in wild‐type N. benthamiana leaves inoculated with C. destructivum () or C. orbiculare (). N. benthamiana leaves inoculated with water () were used as control. The quantity of NbXEGIP1 mRNA was determined relative to the amount of NbEF‐1αmRNA. Means are shown with standard error bars calculated from three or four replications.

Figure 3

Relative RT‐PCR of NbXEGIP1 in wild‐type N. benthamiana plants inoculated with P. syringae pv. tabaci (). N. benthamiana leaves infiltrated with MgCl₂ were used as control (). The quantity of NbXEGIP1 mRNA was determined relative to the amount of NbEF‐1α mRNA. Means are shown with standard error bars calculated from three or four replications.

Figure 4

Relative RT‐PCR of NbXEGIP1 expression in leaves of N. benthamiana (Pto) inoculated with P. syringae pv. tabaci (avrPto) ( ). Controls were leaves infiltrated with MgCl₂ () or P. syringae pv. tabaci containing the vector, pDSK519, without insert (). The quantity of NbXEGIP1 mRNA was determined relative to the amount of NbEF‐1α mRNA. Means are shown with standard error bars calculated from three or four replications.

Figure 5

Effect of virus‐induced gene silencing on expression of NbXEGIP1 in wild‐type N. benthamiana and N. benthamiana (Pto). Relative RT‐PCR of NbXEGIP1 co‐amplified with NbEF‐1α from cDNA of leaves at 96 h post‐inoculation (hpi) by C. destructivum (A) and C. orbiculare (B), at 72 hpi by P. syringae pv. tabaci (C) and 24 hpi by P. syringae pv. tabaci (avrPto) (D). Wild‐type N. benthamiana or N. benthamiana (Pto) were infiltrated with 10 mm MgCl₂ (MgCl₂), A. tumefaciens containing TRV1 and TRV2 with a GFP insert (TRV‐GFP), or A. tumefaciens containing TRV1 and TRV2 with a fragment of NbXEGIP1 (TRV‐NbXEGIP1). After 15–18 days from the TRV treatment to allow for silencing to spread, wild‐type N. benthamiana plants were inoculated with C. destructivum, C. orbiculare or P. syringae pv. tabaci, and N. benthamiana (Pto) plants were inoculated with P. syringae pv. tabaci (avrPto). Lane M is the 100‐bp ladder.

Figure 6

Appearance of youngest fully matured leaf of N. benthamiana at 66 hpi with P. syringae pv. tabaci. Plants had been infiltrated 15–18 days earlier with 10 mm MgCl₂ (MgCl₂ control), A. tumefaciens containing TRV1 and TRV2 with a GFP insert (TRV‐GFP control), or A. tumefaciens containing TRV1 and TRV2 with a fragment of NbXEGIP1 (TRV‐NbXEGIP1).