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, 155 (2), 866-80

The Novel Cyst Nematode Effector Protein 19C07 Interacts With the Arabidopsis Auxin Influx Transporter LAX3 to Control Feeding Site Development

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The Novel Cyst Nematode Effector Protein 19C07 Interacts With the Arabidopsis Auxin Influx Transporter LAX3 to Control Feeding Site Development

Chris Lee et al. Plant Physiol.

Abstract

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.

Figures

Figure 1.
Figure 1.
The cyst nematode effector protein 19C07. A, Sequence comparison of 19C07 orthologs from H. glycines and H. schachtii. 19C07 is a novel 207-amino acid protein containing a putative 21-amino acid secretion signal. 19C07 is highly conserved between these two cyst nematode species containing 99% homologous amino acid sequence. B and C, In situ analysis of 19C07 in parasitic H. glycines cyst nematodes. B, pJ2 nematode. C, pJ4 nematode. DG, Dorsal gland.
Figure 2.
Figure 2.
H. schachtii 19C07 interacts with the Arabidopsis LAX3 auxin influx transporter. A, Yeast two-hybrid analysis. Left column, cotransformants grown on SD-Leu-Trp medium demonstrate that both bait and prey plasmids are present in yeast. Hs19C07 was cloned into the yeast two-hybrid vector pGBKT7, while the original recovered clone (Y.C. 55), the extra N-terminal sequence from Y.C. 55 (Y.C. 55 N-ter), and full-length LAX3 were cloned into pGADT7. pGBKT7 with Hs19C07 was cotransformed with pGADT7 carrying SV40 for a negative control. pGBKT7 with p53 and pGADT7 with SV40 provided a positive control. Blue colonies represent positive interactions in the presence of x-α-gal. Middle and left columns, positive selection of yeast two-hybrid interactions with minimal selection (SD-Leu-Trp-His) and restrictive (SD-Leu-Trp-His-Ade) media. B to I, BiFC confirmation of Hs19C07 interaction with AtLAX3. Bright-field images (B, D, F, and H) and fluorescent images (C, E, G, and I) of onion epidermal cells bombarded with BiFC constructs. B and C, Cobombardment of LAX3 C-eYFP and 19C07 N-eYFP. D and E, Plasmolyzed onion epidermal cell cobombarded with 19C07 N-eYFP and LAX3 C-eYFP. F and G, Cobombardment of LAX3 C-eYFP and 4D09 N-eYFP. H and I, Cobombardment of 19C07 N-eYFP and NHL3 C-eYFP.
Figure 3.
Figure 3.
LAX3 and a LAX3-inducible gene are expressed in and surrounding developing syncytia. A and B, LAX3pro:GUS Arabidopsis roots infected with H. schachtii pJ2 (A) and pJ3 (B). C to E, PG expression demonstrated with PGpro:GUS Arabidopsis in uninfected roots. PG is expressed in cell layers overlaying developing lateral root primordia (C), emerging lateral roots (D), and in the cells shortly after the lateral root has emerged (E). F and G, PGpro:GUS Arabidopsis roots infected with H. schachtii. F, pJ2. G, Syncytium (Syn) at 8 dpi. Bars = 100 μm.
Figure 4.
Figure 4.
LAX3 is induced early in developing feeding sites and expressed in cells to be incorporated into syncytia. Arabidopsis lax3 and LAX3-YFP lines were observed before and after infection. A, C, E, G, I, K, M, and O, Bright-field images. B, D, F, H, J, L, N, and P, Fluorescent images. A and B, LAX3-YFP expression in the cells directly above an emerging lateral root primordium. C and D, LAX3-YFP expression persists in the cells above the emerged lateral root. E and F, H. schachtii-infected root 2 dpi with LAX3-YFP expression initiated in the feeding site as well as in several cells surrounding the developing feeding site. Blue N is on the infecting nematode. G and H, The same root infection site from E and F at 3 dpi. The LAX3-YFP signal has spread about 1 mm from the original infection site. Red arrows point to the nematode head. I to P, LAX3-YFP expression in the same syncytium at 3 dpi. Blue arrows point to the nematode (N), and red arrows point to the syncytium. Times are as follows: 3 dpi (I and J), 4 dpi (K and L), 5 dpi (M and N), and 7 dpi (O and P). Bars = 100 μm.
Figure 5.
Figure 5.
Auxin influx transport mutants decrease nematode infectivity. A, At least 30 roots from Arabidopsis Col-0, aux1lax3, and aux1lax1lax2lax3 infected with H. schachtii were compared for penetration 48 h after infection. Means were not significantly different from the wild type using a two-tailed t test assuming unequal variances. Error bars indicate se. For aux1lax3, P = 0.901; for aux1lax1lax2lax3, P = 0.414 B, Three replicates of 36 individuals from Col-0 and lax3 lines were infected on 12-well plates with 250 infective J2 of H. schachtii. pJ4 female nematodes were counted per plant at 14 dpi, and adult female nematodes were counted at 30 dpi. Error bars indicate se. C, Three replicates of 36 individuals from Col-0 and aux1 lines were infected on 12-well plates with 250 infective J2 of H. schachtii. pJ4 female nematodes were counted per plant at 14 dpi, and adult female nematodes were counted at 30 dpi. Error bars indicate se. D, Three replicates of at least four vertical plates with eight individuals of Col-0, aux1lax3, and aux1lax1lax2lax3 genotypes were infected with H. schachtii. pJ4 female nematodes were counted per plate at 14 dpi, and adult female nematodes were counted at 30 dpi. Error bars indicate se. * P < 0.05 using a two-tailed t test.
Figure 6.
Figure 6.
Nematode infection induces lateral root formation in the aux1lax3 double and aux1lax1lax2lax3 quadruple mutants. A, Col-0, lax3, aux1lax3, and aux1lax1lax2lax3 seedlings either uninfected or infected (5 dpi) grown on vertical plates. Insets show infected aux1lax3 and aux1lax1lax2lax3 mutant lines. Arrowheads point to infection sites. Edge of squares = 1.3 cm. B, Lateral roots emerging from syncytia in infected aux1lax3 and aux1lax1lax2lax3 roots at 11 dpi. C, Lateral roots from all feeding sites were counted on three replicates of at least two vertical plates sown with six individuals from each line. Error bars indicate se. Letter grouping above bars indicates significance groups (P < 0.05). Plant lines that share a letter are not significantly different from each other using a two-tailed t test.
Figure 7.
Figure 7.
Ectopic expression of 19C07 in Arabidopsis reduces infectivity of H. schachtii. A, Relative expression assayed by qRT-PCR of Hs19C07 in four independent homozygous Arabidopsis overexpression lines. Transcript levels were compared with Col-0 and are expressed in a logarithmic scale. Error bars indicate 95% confidence intervals. The bottom shows RT-PCR analysis of the coding region of the 19C07 transgene in the lines tested. B, Three replicates of 36 individuals from Col-0 and 19C07 overexpression lines were infected on 12-well plates with 250 infective J2 of H. schachtii. pJ4 female nematodes were counted on individuals at 14 dpi, and adult female nematodes were counted at 30 dpi. Error bars indicate se. * = P < 0.05.
Figure 8.
Figure 8.
Ectopic expression of 19C07 in Arabidopsis leads to earlier lateral root emergence. A and B, Initial lateral root emergence of lax3 and LAX3-YFP (A) and lax3 and LAX3-YFP × 19C07 6-13-3 overexpression line 5 d after seeding (T = 0) through 8 d after seeding (T = 72 h). Bars = 100 μm. C, The number of LAX3-YFP sites of expression on 7- and 11-d-old seedlings. Lateral root initiation sites were counted on two replicates of two vertical plates seeded with six individuals for each line. No significant difference was found using a two-tailed t test. Error bars indicate se.

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