Fig 1.
Host root exudates stimulate a stylet thrusting response in P. coffeae.
Root exudates from the hosts banana, carrot, coffee, maize and potato were evaluated for their effects on the rate of stylet thrusting in individuals of mixed life stages (A) and the proportion of individuals of mixed life stages thrusting their stylets (B). H2O and 5 mM 5-hydroxytryptamine (5-HT) were used as negative and positive controls, respectively. Values are means ± SEM (n = 30 with different letters indicating significant differences between treatments P<0.01 (One-way ANOVA, SNK test).
Fig 2.
Pc-eng-1 and Pc-xyl are expressed in the pharyngeal gland cells throughout nematode development.
Schematic representation of a plant-parasitic nematode (A) outlines the pharyngeal gland cells where the digoxigenin-labelled probes of Pc-eng-1 (B) and Pc-xyl (C) are localised. The median bulb is indicated for reference (arrow). No corresponding staining occurred when the negative control probes were used (D and E). Scale = 50 μm. Analysis by qRT-PCR confirmed that Pc-eng-1 and Pc-xyl are both expressed at egg, juvenile (juv), female and male life stages (F). Both genes showed significantly increased expression as the nematode developed. Expression was normalised to Elongation Factor and presented relative to expression in eggs. Values are means ± SEM (n = 3 pools of individuals) with different letters indicating significant differences between treatments P<0.05 (One-way ANOVA, SNK test).
Fig 3.
Expression of Pc-eng-1 and Pc-xyl is influenced by the host plant.
Relative expression of Pc-eng-1 and Pc-xyl significantly differed between mixed life stage pools of P. coffeae recovered from host roots of banana, carrot, coffee, maize or potato (A). Values are means ± SEM (n = 8 pools of mixed stages) shown relative to expression in nematodes recovered from the roots of potato plants. Exposure of nematodes extracted from the same host (carrot) to different root exudates after 48 h in water induced expression of Pc-eng-1 and Pc-xyl. The expression profiles were broadly similar to those observed when nematodes were extracted directly from the different host roots. (B). Values are means ± SEM (n = 4 pools of mixed stages) shown relative to expression in nematodes maintained in water. Different letters indicate significant differences between treatments P<0.05 (One-way ANOVA, SNK test).
Fig 4.
Pc-eng-1 and Pc-xyl expression correlates with cellulose and xylan quantities exuded by plant roots.
There was a linear regression between the quantity of cellulose and xylan in the exudates (n = 5) and the expression level of Pc-eng-1 (A) (P<0.05, R2 = 0.89) and Pc-xyl (B) (P<0.01. R2 = 0.988) (n = 4 pools of mixed stages), respectively. Exudates from the roots of Arabidopsis rsw1 and glz1 were confirmed to be deficient in cellulose and xylan, respectively compared to Col-0 (WT) (C and D). Cellulose and xylan deficiencies caused significantly lower induction of Pc-eng-1 and Pc-xyl expression in P. coffeae after 6 h exposure (E). Pc-pel remained unaffected by the changes in exudate whilst exudates from the roots of mur3 had no effect on expression of any gene studied. Values are means ± SEM (n = 4 pools of mixed stage P. coffeae) with different letters indicating significant differences between treatments P<0.05 (One-way ANOVA, SNK test). Gene expression levels (A, B and E) are presented relative to expression in water.
Fig 5.
Cellulose and xylan specifically upregulate expression of Pc-eng-1 and Pc-xyl, respectively.
Polynomial regression analysis establishes a significant increase in expression of Pc-eng-1 (black circle) and Pc-xyl (black square) in P. coffeae with cellulose (A) and xylan (B) concentrations respectively after 6 h exposure (P< 0.01, R2 = 0.96 and P<0.001, R2 = 0.98, respectively). Expression of Pc-pel (white triangle) remained unaffected. Values are means ± SEM (n = 4 pools of mixed stage P. coffeae) and relative to expression in water.
Fig 6.
RNA interference of Pc-eng-1 and Pc-xyl in P. coffeae reduces infection of potato and maize roots.
The dsRNA molecules reduced expression of their respective targets whilst gfp dsRNA (a gene that is absent from the nematode) had no effect (A and B). Expression is presented relative to that for control nematodes incubated in buffer only. Pc-eng-1 dsRNA and Pc-xyl dsRNA reduced the infection of P. coffeae in both potato and maize root systems (C). Values are means ± SEM (n = 6 pools of mixed stage P. coffeae) with different letters denoting significant differences between treatments P<0.05 (One-way ANOVA, SNK test).