JC 13.04.2017

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Slide 1

Journal Club April 13, 2017 MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction Kelliher et al., Nature, 2017

Slide 2

Double fertilization Synergids and egg Haploid gametophytes can be induced to develop as doubled haploid sporophytes through in vitro tissue culture or haploid seed induction. In maize, haploids are routinely produced using crosses to Stock 6, reported in 1959 as producing 2–3% maternal haploid seed during self-pollination or when outcrossed as a male.

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Clarify the developmental genetics underlying haploid induction Stock 6 derivative RWK (approximately 13% HIR*) was crossed to the inbreds NP2460 and NP2391, and subsequently backcrossed to RWK to generate mapping populations. * haploid induction rate (HIR) plant and 1 cM = cross-over occurs in 1/100 cases - Through map-based cloning

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Map-based cloning Stock 6 RWK NP2460 & NP2391 BC to RWK 0.57 Mb missing (PGM) frame shift

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A screen of 19 Stock 6 derivatives found that all 19 were homozygous for the inducer haplotypes of these two candidates, including NP2222-Haploid Inducer (NP2222-HI) Heterologous complementation of NP2222-HI (10.2% HIR) with wild-type copies of those genes virtually eliminated haploid induction and kernel abortion for GRMZM2G471240 only Because GRMZM2G062320 did not complement, it probably has no role in haploid induction. We renamed GRMZM2G471240 MATRILINEAL (MTL) and the mutant allele matrilineal (mtl).

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New alleles for mtl frame-shift mutations in MTL MTLTAL-FS HIR of 4.0 – 12.5%

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Ploidy status confirmed Flow cytometry DAPI-stained cells The more signal, the more DNA (i.e., 1n vs. 2n)

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Mtl is gametophyte-specific The FS in mtl @ a.a. 380 STOP MTL expression level

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MTL localization The FS in mtl compromises MTL localization or stability in haploid inducer pollen. mtl pollen mtl pollen MTL pollen NP2222 O + NP2222 O + NP2222 O +

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Pollen germination and tube growth are normal

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The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. NLD is the key factor triggering gynogenesis in maize after pollination by inducer lines.

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Arabidopsis* root tips expressing either wild-type NLD (A, B) or truncated NLD-PK6 (C, D) fused to citrine fluorescent protein. *Transgenic Arabidopsis Kelliher et al., 2017

Slide 13

Diffuse signal in the cytoplasm in case of mtl Kelliher et al., 2017 It’s not background exine fluorescence

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NLD (a PL) Phospholipases (PLs) are enzymes that cleave phospholipids, the major component of all cellular membranes. (i) NLD promoter strongly active in mature pollen and pollen tubes, (ii) NLD protein found in sperm cell plasma membranes, (iii) A 4-bp insertion promotes the formation of haploid embryos, (iv) Localization to the plasma membrane is lost, and (v) Its annotation as a patatin-like phospholipase A suggests that NLD is implicated in membrane lipid homeostasis of the sperm cells.

Slide 15

This allele is a rare mutation - after maize domestication, since it was not detected in the teosinte accessions.

Summary: Journal Club meeting, April 13, 2017

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