Three-dimensional imaging reveals that positions of cyst nematode feeding sites relative to xylem vessels differ between susceptible and resistant wheat.

KEY MESSAGE: Resistance conferred by the Cre8 locus of wheat prevents cereal cyst nematode feeding sites from reaching and invading root metaxylem vessels. Cyst nematodes develop syncytial feeding sites within plant roots. The success of these sites is affected by host plant resistance. In wheat (Triticum aestivum L.), 'Cre' loci affect resistance against the cereal cyst nematode (CCN) Heterodera avenae. To investigate how one of these loci (Cre8, on chromosome 6B) confers resistance, CCN-infected root tissue from susceptible (-Cre8) and resistant (+Cre8) wheat plants was examined using confocal microscopy and laser ablation tomography. Confocal analysis of transverse sections showed that feeding sites in the roots of -Cre8 plants were always adjacent to metaxylem vessels, contained many intricate 'web-like' cell walls, and sometimes 'invaded' metaxylem vessels. In contrast, feeding sites in the roots of +Cre8 plants were usually not directly adjacent to metaxylem vessels, had few inner cell walls and did not 'invade' metaxylem vessels. Models based on data from laser ablation tomography confirmed these observations. Confocal analysis of longitudinal sections revealed that CCN-induced xylem modification that had previously been reported for susceptible (-Cre8) wheat plants is less extreme in resistant (+Cre8) plants. Application of a lignin-specific stain revealed that secondary thickening around xylem vessels in CCN-infected roots was greater in +Cre8 plants than in -Cre8 plants. Collectively, these results indicate that Cre8 resistance in wheat acts by preventing cyst nematode feeding sites from reaching and invading root metaxylem vessels.

Transporters involved in pH and K+ homeostasis affect pollen wall formation, male fertility, and embryo development.

Flowering plant genomes encode multiple cation/H+ exchangers (CHXs) whose functions are largely unknown. AtCHX17, AtCHX18, and AtCHX19 are membrane transporters that modulate K+ and pH homeostasis and are localized in the dynamic endomembrane system. Loss of function reduced seed set, but the particular phase(s) of reproduction affected was not determined. Pollen tube growth and ovule targeting of chx17chx18chx19 mutant pollen appeared normal, but reciprocal cross experiments indicate a largely male defect. Although triple mutant pollen tubes reach ovules of a wild-type pistil and a synergid cell degenerated, half of those ovules were unfertilized or showed fertilization of the egg or central cell, but not both female gametes. Fertility could be partially compromised by impaired pollen tube and/or sperm function as CHX19 and CHX18 are expressed in the pollen tube and sperm cell, respectively. When fertilization was successful in self-pollinated mutants, early embryo formation was retarded compared with embryos from wild-type ovules receiving mutant pollen. Thus CHX17 and CHX18 proteins may promote embryo development possibly through the endosperm where these genes are expressed. The reticulate pattern of the pollen wall was disorganized in triple mutants, indicating perturbation of wall formation during male gametophyte development. As pH and cation homeostasis mediated by AtCHX17 affect membrane trafficking and cargo delivery, these results suggest that male fertility, sperm function, and embryo development are dependent on proper cargo sorting and secretion that remodel cell walls, plasma membranes, and extracellular factors.

MicroRNA-resistant alleles of HOMEOBOX DOMAIN-2 modify inflorescence branching and increase grain protein content of wheat.

Plant and inflorescence architecture determine the yield potential of crops. Breeders have harnessed natural diversity for inflorescence architecture to improve yields, and induced genetic variation could provide further gains. Wheat is a vital source of protein and calories; however, little is known about the genes that regulate the development of its inflorescence. Here, we report the identification of semidominant alleles for a class III homeodomain-leucine zipper transcription factor, HOMEOBOX DOMAIN-2 (HB-2), on wheat A and D subgenomes, which generate more flower-bearing spikelets and enhance grain protein content. These alleles increase HB-2 expression by disrupting a microRNA 165/166 complementary site with conserved roles in plants; higher HB-2 expression is associated with modified leaf and vascular development and increased amino acid supply to the inflorescence during grain development. These findings enhance our understanding of genes that control wheat inflorescence development and introduce an approach to improve the nutritional quality of grain.

Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant 'Kushen' (Sophora flavescens Ait.).

Kushen root, from the woody legume Sophora flavescens, is a traditional Chinese medicine that is a key ingredient in several promising cancer treatments. This activity is attributed in part to two quinolizidine alkaloids (QAs), oxymatrine and matrine, that have a variety of therapeutic activities in vitro. Genetic selection is needed to adapt S. flavescens for cultivation and to improve productivity and product quality. Genetic diversity of S. flavescens was investigated using genotyping-by-sequencing (GBS) on 85 plants grown from seeds collected from 9 provinces of China. DArTSeq provided over 10,000 single nucleotide polymorphism (SNP) markers, 1636 of which were used in phylogenetic analysis to reveal clear regional differences for S. flavescens. One accession from each region was selected for PCR-sequencing to identify gene-specific SNPs in the first two QA pathway genes, lysine decarboxylase (LDC) and copper amine oxidase (CAO). To obtain SfCAO sequence for primer design we used a targeted transcript capture and assembly strategy using publicly available RNA sequencing data. Partial gene sequence analysis of SfCAO revealed two recently duplicated genes, SfCAO1 and SfCAO2, in contrast to the single gene found in the QA-producing legume Lupinus angustifolius. We demonstrate high efficiency converting SNPs to Kompetitive Allele Specific PCR (KASP) markers developing 27 new KASP markers, 17 from DArTSeq data, 7 for SfLDC, and 3 for SfCAO1. To complement this genetic diversity analysis a field trial site has been established in South Australia, providing access to diverse S. flavescens material for morphological, transcriptomic, and QA metabolite analysis. Analysis of dissected flower buds revealed that anthesis occurs before buds fully open suggesting a potential for S. flavescens to be an inbreeding species, however this is not supported by the relatively high level of heterozygosity observed. Two plants from the field trial site were analysed by quantitative real-time PCR and levels of matrine and oxymatrine were assessed in a variety of tissues. We are now in a strong position to select diverse plants for crosses to accelerate the process of genetic selection needed to adapt kushen to cultivation and improve productivity and product quality.

Infection by cyst nematodes induces rapid remodelling of developing xylem vessels in wheat roots.

Cyst nematodes induce host-plant root cells to form syncytia from which the nematodes feed. Comprehensive histological investigation of these feeding sites is complicated by their variable shape and their positions deep within root tissue. Using tissue clearing and confocal microscopy, we examined thick (up to 150 µm) sections of wheat roots infected by cereal cyst nematodes (Heterodera avenae). This approach provided clear views of feeding sites and surrounding tissues, with resolution sufficient to reveal spatial relationships among nematodes, syncytia and host vascular tissues at the cellular level. Regions of metaxylem vessels near syncytia were found to have deviated from classical developmental patterns. Xylem vessel elements in these regions had failed to elongate but had undergone radial expansion, becoming short and plump rather than long and cylindrical. Further investigation revealed that vessel elements cease to elongate shortly after infection and that they later experience delays in secondary thickening (lignification) of their outer cell walls. Some of these elements were eventually incorporated into syncytial feeding sites. By interfering with a developmental program that normally leads to programmed cell death, H. avenae may permit xylem vessel elements to remain alive for later exploitation by the parasite.