Internuclear Gene Silencing

A puzzling case of gene silencing was discovered in the potato pathogen Phytophthora infestans (Straminipila) that is maintained in the progeny nuclei even in the absence of the transgene (van West et al., 1999). The inf-1 gene encodes a secretory protein called elicitin—a hydrophobin that induces necrosis in the plant, thereby restricting the spread of pathogenic mycelium. By transforming P. infestans with the plasmid-containing inf-1 gene, mutants were produced that were silenced in the production of elicitin. The protoplasts of silenced and non-silenced strains were fused to obtain a heterokaryotic (silenced) strain that was resolved into homokaryotic component strains by nuclear separation using the uninucleate zoospores of this "fungus" (see Appendix for figure). The individual nuclear types multiplied mitotically in the regenerating mycelium. The homokaryotic strains produced from uninuclear zoospores were silenced, i.e., once gene silencing was induced, it was maintained in the homokaryotic strain even in the absence of the transgene. Though both internuclear gene silencing (IGS) and quelling are dominant, IGS differs from quelling in being infectious, i.e., transmitted from nuclei to nuclei, whereas quelling is not transmitted. Moreover, where the presence of transgene is essential for quelling, it is not required for IGS (Figure 9.10).

Neurospora crassa: Quelling of al-1 is dominant and reversible.

Neurospora crassa: Silencing of heterologous hph is recessive and reversible.

Phytophthora infestans: silencing of inf-1 is dominant and infectious.

Orange, non-silenced j albino, silenced

Heterokaryon, silenced (albino)

Nuclear separation

Orange Orange

(Non-silenced) (Non-silenced)

Silenced hph gene Non-silenced hph gene

Hygs) I

(al-1 +; hph+; Hygs + al-1; hph+; HygR) Heterokaryon, non-silenced

Nuclear separation

(Non-silenced) (Non-silenced)

Wild type inf-1 i inf-1 gene duplication non-silenced 1 silenced

Heterokaryon, silenced

Nuclear separation f \

Wild type inf-1 Duplicated inf-1

silenced (silenced)

Figure 9.10 Transgene silencing in heterokaryons of Neurospora crassa and Phytophthora infestans. Adapted from Dev and Maheshwari (2003).

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