Analysis of Putative Apoplastic Effectors from the Nematode, <i>Globodera rostochiensis</i>, and Identification of an Expansin-Like Protein That Can Induce and Suppress Host Defenses

<div><p>The potato cyst nematode, <i>Globodera rostochiensis</i>, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative <i>G. rostochiensis</i> putative apoplastic effectors in vectors for expression <i>in planta</i>. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (<i>Gr</i>UBCEP12) and an expansin-like protein (<i>Gr</i>EXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. <i>Gr</i>EXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.</p></div

<i>Gr</i>EXPB2 and <i>Gr</i>UBCEP12 proteins suppress virus resistance mediated by the <i>N</i> gene.

<p><i>N. benthamiana</i> leaves were co-infiltrated with <i>Agrobacterium</i> carrying binary vectors expressing PVX-GFP, N and P50 together with pEAQ35S expressing (a) 1, <i>Gr</i>UBCEP12; 2, empty vector; (b) 1, <i>Gr</i>EXPB2; 2, empty vector; 3, P0; 4, P38. GFP expression was visualized under UV illumination at 4 DPI. (c-d) Anti GFP immune blotting was performed on total protein samples taken at 4 DPI from <i>N. benthamiana</i> leaf patches co-expressing the combinations of constructs described in a and b. The number on the blot corresponds to the number on the leaf above each blot. Ponceau S staining (lower panel) was used to show equal loading. </p

<i>Gr</i>UBCEP12 and <i>Gr</i>SKP1 alter plant morphology.

<p><i>N. benthamiana</i> plants were infected by agroinfiltration with (a) empty PVX vector (EV), (b) PVX-<i>Gr</i>SKP1, (c) PVX-<i>Gr</i>UBCEP12, or (d) PVX-<i>Gr</i>UBCEP12ΔSP. Photographs were taken at 16 DPI. Systemic expression in potato of (e) PVX and (f) PVX-<i>Gr</i>UBCEP12ΔSP in potato cultivar Katahdin. Photographs were taken at 30 DPI. </p

CLE peptides induce dramatic phenotypes in <i>N. benthamiana</i>.

<p><i>N. benthamiana</i> plants were infected by agroinfiltration with (a) empty PVX vector (EV), (b) PVX-<i>Gr</i>CLE1, (c) PVX-<i>Gr</i>CLE1ΔSP, (d) PVX-<i>Gr</i>CLE4B, (e) PVX-<i>Gr</i>CLE4BΔSP and (f) PVX-<i>Gr</i>CLE4D. Photographs were taken at 21 DPI. </p

Description of cloned apoplastic secreted effector proteins.

<p>Description of cloned apoplastic secreted effector proteins.</p

<i>Gr</i>EXPB2 and <i>Gr</i>UBCEP12 suppress Rx-mediated resistance to PVX.

<p><i>N. benthamiana</i> leaves were co-infiltrated with <i>Agrobacterium</i> carrying binary vectors expressing PVX-GFP and 35S-Rx together with pEAQ35S vectors expressing 1, <i>Gr</i>EXPB2; 2, empty vector; 3, <i>Gr</i>SPRYSEC-19ΔSP; 4, Rx replaced with empty vector; 5, <i>Gr</i>UBCEP12; 6, empty vector. (a) GFP expression was visualized and photographed under UV illumination at 4 DPI. (b) Anti-GFP immune blotting was performed on total protein samples taken at 4 DPI from <i>N. benthamiana</i> patches expressing the same construct combinations as described above. Ponceau S staining (lower panel) was used to show equal loading. </p

Cloning and expression strategy for putative <i>G. rostochiensis</i> effector proteins.

<p>Cloning and functional analysis of candidate secreted effector proteins <i>in planta</i> included; (i) PCR amplification of effector encoding genes from <i>G. rostochiensis</i> cDNA with their cognate SP (red); (ii) Cloning into the donor vector pDONR207 or pDONR221; (iii) Sequencing of multiple clones for sequence confirmation; (iv) Transfer of cDNAs from donor vector to gateway compatible pEAQ35S, PVX and PVX-HB, where ^§ is Gateway compatible pGR106 and ^¶ is Gateway compatible pGR103; (v) <i>In planta</i> expression of effectors by agroinfiltration and agroinfection; (vi) Identification of phenotype induced in different solanaceous plants and assessment for cell death suppression. </p

<i>Gr</i>PEL1, <i>Gr</i>PEL2 and <i>Gr</i>MTP induce chlorosis and necrosis in <i>N. benthamiana</i> and tomato.

<p><i>N. benthamiana</i> plants were infected by agroinfiltration with (a) PVX-GFP, (b) PVX-<i>Gr</i>PEL1, (c) PVX-<i>Gr</i>PEL2, (d) PVX-<i>Gr</i>MTP. Plants were photographed at 21 DPI. Tomato plants, cultivar Starfire, were inoculated with (e) PVX-<i>Gr</i>PEL1 and (f) PVX-<i>Gr</i>PEL2 or with PVX-GFP (e, f, right hand side). Plants were photographed at 28 DPI. </p

<i>Gr</i>EXPB2 induces necrosis in tomato and potato and chlorosis and dwarfing in <i>N. benthamiana</i>.

<p><i>N. benthamiana</i> infected with (a) PVX-<i>Gr</i>EXPB2 clone 1A (top) or empty PVX vector (EV) (bottom). (b) PVX-<i>Gr</i>EXPB2ΔSP clone 1A, (c) PVX-<i>Gr</i>EXPB2:HIS-HA clone 12b, (d) PVX-<i>Gr</i>EXPB2:HIS-HA clone 7g, (e) PVX-<i>Gp</i>EXPB2:HIS-HA clone 15l. Plants were photographed at 20 DPI. Tomato cultivars (f, k, l) LA 1792 and (g) Cherry, were tooth pick inoculated with PVX derivatives or tooth pick only. The numbers correspond to 1, <i>Gr</i>EXPB2 clone 1A; 2, <i>Gr</i>EXPB2:HIS-HA clone 1A; 3, tooth pick only; 4, <i>Gr</i>EXPB2ΔSP clone 1A; 5, EXPB1:HIS-HA; 6, GFP; 7, <i>Gr</i>PEL1; 8, <i>Gr</i>EXPB2:HIS-HA clone 12b; 9, <i>Gp</i>EXPB2:HIS-HA clone 15l; 10, <i>Gr</i>EXPB2:HIS-HA clone 22j; 11, <i>Gr</i>EXPB2:HIS-HA clone 5a; 12, <i>Gr</i>EXPB2:HIS-HA clone 6b; and 13, <i>Gr</i>EXPB2:HIS-HA clone 7g. Leaves were photographed at 10 DPI. Tomato cultivars (h) LA 1792 and (i) Moneymaker were agroinfiltrated with pEAQ35S constructs expressing 1, <i>Gr</i>EXPB2 clone 1A; 2, AtRx and 4, Empty vector. Leaves were photographed at 6 DPI. Potato cultivar (j) Green Mountain was tooth pick inoculated with PVX constructs as in (f, g, k, l). Leaves were photographed at 15 DPI. (m) HA-tagged versions of EXPB2 variants, as indicated, were expressed in <i>N. benthamiana</i> leaves from the PVX vector and total protein extracts were prepared from infiltrated patches 4 DPI, followed by Anti HA immune blotting. Ponceau S staining (lower panel) was used to show equal loading. </p