Sensitivity of jarrah (Eucalyptus marginata) to phosphate, phosphite, and arsenate pulses as influenced by fungal symbiotic associations.

Many plant species adapted to P-impoverished soils, including jarrah (Eucalyptus marginata), develop toxicity symptoms when exposed to high doses of phosphate (Pi) and its analogs such as phosphite (Phi) and arsenate (AsV). The present study was undertaken to investigate the effects of fungal symbionts Scutellospora calospora, Scleroderma sp., and Austroboletus occidentalis on the response of jarrah to highly toxic pulses (1.5 mmol kg(-1) soil) of Pi, Phi, and AsV. S. calospora formed an arbuscular mycorrhizal (AM) symbiosis while both Scleroderma sp. and A. occidentalis established a non-colonizing symbiosis with jarrah plants. All these interactions significantly improved jarrah growth and Pi uptake under P-limiting conditions. The AM fungal colonization naturally declines in AM-eucalypt symbioses after 2-3 months; however, in the present study, the high Pi pulse inhibited the decline of AM fungal colonization in jarrah. Four weeks after exposure to the Pi pulse, plants inoculated with S. calospora had significantly lower toxicity symptoms compared to non-mycorrhizal (NM) plants, and all fungal treatments induced tolerance against Phi toxicity in jarrah. However, no tolerance was observed for AsV-treated plants even though all inoculated plants had significantly lower shoot As concentrations than the NM plants. The transcript profile of five jarrah high-affinity phosphate transporter (PHT1 family) genes in roots was not altered in response to any of the fungal species tested. Interestingly, plants exposed to high Pi supplies for 1 day did not have reduced transcript levels for any of the five PHT1 genes in roots, and transcript abundance of four PHT1 genes actually increased. It is therefore suggested that jarrah, and perhaps other P-sensitive perennial species, respond positively to Pi available in the soil solution through increasing rather than decreasing the expression of selected PHT1 genes. Furthermore, Scleroderma sp. can be considered as a fungus with dual functional capacity capable of forming both ectomycorrhizal and non-colonizing associations, where both pathways are always accompanied by evident growth and nutritional benefits.

A novel plant-fungus symbiosis benefits the host without forming mycorrhizal structures.

• Most terrestrial plants form mutually beneficial symbioses with specific soil-borne fungi known as mycorrhiza. In a typical mycorrhizal association, fungal hyphae colonize plant roots, explore the soil beyond the rhizosphere and provide host plants with nutrients that might be chemically or physically inaccessible to root systems. • Here, we combined nutritional, radioisotopic ((33)P) and genetic approaches to describe a plant growth promoting symbiosis between the basidiomycete fungus Austroboletus occidentalis and jarrah (Eucalyptus marginata), which has quite different characteristics. • We show that the fungal partner does not colonize plant roots; hyphae are localized to the rhizosphere soil and vicinity and consequently do not transfer nutrients located beyond the rhizosphere. Transcript profiling of two high-affinity phosphate (Pi) transporter genes (EmPHT1;1 and EmPHT1;2) and hyphal-mediated (33)Pi uptake suggest that the Pi uptake shifts from an epidermal to a hyphal pathway in ectomycorrhizal plants (Scleroderma sp.), similar to arbuscular mycorrhizal symbioses, whereas A. occidentalis benefits its host indirectly. The enhanced rhizosphere carboxylates are linked to growth and nutritional benefits in the novel symbiosis. • This work is a starting point for detailed mechanistic studies on other basidiomycete-woody plant relationships, where a continuum between heterotrophic rhizosphere fungi and plant beneficial symbioses is likely to exist.

Ecto- and arbuscular mycorrhizal symbiosis can induce tolerance to toxic pulses of phosphorus in jarrah (Eucalyptus marginata) seedlings.

In common with many plants native to low P soils, jarrah (Eucalyptus marginata) develops toxicity symptoms upon exposure to elevated phosphorus (P). Jarrah plants can establish arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) associations, along with a non-colonizing symbiosis described recently. AM colonization is known to influence the pattern of expression of genes required for P uptake of host plants and our aim was to investigate this phenomenon in relation to P sensitivity. Therefore, we examined the effect on hosts of the presence of AM and ECM fungi in combination with toxic pulses of P and assessed possible correlations between the induced tolerance and the shoot P concentration. The P transport dynamics of AM (Rhizophagus irregularis and Scutellospora calospora), ECM (Scleroderma sp.), non-colonizing symbiosis (Austroboletus occidentalis), dual mycorrhizal (R. irregularis and Scleroderma sp.), and non-mycorrhizal (NM) seedlings were monitored following two pulses of P. The ECM and A. occidentalis associations significantly enhanced the shoot P content of jarrah plants growing under P-deficient conditions. In addition, S. calospora, A. occidentalis, and Scleroderma sp. all stimulated plant growth significantly. All inoculated plants had significantly lower phytotoxicity symptoms compared to NM controls 7 days after addition of an elevated P dose (30 mg P kg(-1) soil). Following exposure to toxicity-inducing levels of P, the shoot P concentration was significantly lower in R. irregularis-inoculated and dually inoculated plants compared to NM controls. Although all inoculated plants had reduced toxicity symptoms and there was a positive linear relationship between rank and shoot P concentration, the protective effect was not necessarily explained by the type of fungal association or the extent of mycorrhizal colonization.

The reduced mycorrhizal colonisation (rmc) mutation of tomato disrupts five gene sequences including the CYCLOPS/IPD3 homologue.

Arbuscular mycorrhizal (AM) symbiosis in vascular plant roots is an ancient mutualistic interaction that evolved with land plants. More recently evolved root mutualisms have recruited components of the AM signalling pathway as identified with molecular approaches in model legume research. Earlier we reported that the reduced mycorrhizal colonisation (rmc) mutation of tomato mapped to chromosome 8. Here we report additional functional characterisation of the rmc mutation using genotype grafts and proteomic and transcriptomic analyses. Our results led to identification of the precise genome location of the Rmc locus from which we identified the mutation by sequencing. The rmc phenotype results from a deletion that disrupts five predicted gene sequences, one of which has close sequence match to the CYCLOPS/IPD3 gene identified in legumes as an essential intracellular regulator of both AM and rhizobial symbioses. Identification of two other genes not located at the rmc locus but with altered expression in the rmc genotype is also described. Possible roles of the other four disrupted genes in the deleted region are discussed. Our results support the identification of CYCLOPS/IPD3 in legumes and rice as a key gene required for AM symbiosis. The extensive characterisation of rmc in comparison with its 'parent' 76R, which has a normal mycorrhizal phenotype, has validated these lines as an important comparative model for glasshouse and field studies of AM and non-mycorrhizal plants with respect to plant competition and microbial interactions with vascular plant roots.

Significant reduction of fungal disease symptoms in transgenic lupin (Lupinus angustifolius) expressing the anti-apoptotic baculovirus gene p35.

Narrow-leafed lupin (NLL; Lupinus angustifolius) is a recently domesticated but anciently propagated crop with significant value in rotation with cereals in Mediterranean climates. However, several fungal pathogens, traditionally termed necrotrophs, severely affect broad-acre production and there is limited genetic resistance in the NLL germplasm pool. Symptoms of many of these diseases appear as localized areas of dead cells exhibiting markers of programmed cell death. Based on our previous research, we hypothesized that engineered expression of the baculovirus anti-apoptotic p35 gene might reduce symptoms of these diseases. Using Agrobacterium tumefaciens-mediated transformation of a cultivar highly susceptible to several pathogens, 14 independent NLL lines containing both the p35 and bar genes were obtained (p35-NLL). Integration and expression of the transgenes were confirmed by polymerase chain reaction (PCR), progeny testing, Southern blot, Northern blot and reverse transcriptase-PCR analyses. Fecundity and nodulation were not altered in these lines. Third or fourth generation p35-NLL lines were challenged with necrotrophic fungal pathogens (anthracnose in stem and leaf, and Pleiochaeta root rot and leaf brown spot) in controlled environment conditions. Several p35-NLL lines had significantly reduced disease symptoms. Interestingly, as with natural resistance, no single line was improved for all three diseases which possibly reflecting spatial variation of p35 expression in planta. These data support an alternative molecular definition for 'necrotrophic disease' in plants and suggest new routes for achieving resistance against a range of pathogens.

Multiple genetic loci for zinc uptake and distribution in barley (Hordeum vulgare).

Micronutrient malnutrition, often called 'hidden hunger', affects over two billion people globally. This is particularly problematic in developing countries where widespread zinc (Zn) deficiency exists as a result of a predominantly plant-based diet. Furthermore, supplemental fertilizers are often unavailable or unaffordable in impoverished regions where soil infertility is common. Delivery of more Zn via food grains is theoretically possible through selective breeding strategies, but severe technical difficulties associated with trace element research have limited research on the underlying genetic components of Zn nutrition. Genetic dissection of Zn nutrition involved a pre-existing doubled haploid mapping population of barley (Hordeum vulgare). Association of mineral nutrient accumulation traits with regions of the barley genome was determined in two seasons of growth to maturity, using mapmanager qtx and QGene 4.0. Nine genetic loci segregating in the population associated clearly with measured traits, including five that contributed to grain Zn status. Pooling two-row doubled haploids by selecting the three most favourable alleles increased grain Zn content and concentration by an average of 53 and 75%, respectively. These results will inform breeding efforts for increased Zn density in the major food grain, wheat (Triticum aestivum), by enabling syntenic marker-assisted selection in conventional breeding programmes.

Leptosphaeria maculans elicits apoptosis coincident with leaf lesion formation and hyphal advance in Brassica napus.

Programmed cell death, with many of the morphological markers of apoptosis, is increasingly recognized as an important process in plant disease. We have investigated the involvement and potential role of apoptosis during the formation of leaf lesions by the fungus Leptosphaeria maculans on susceptible Brassica napus cv. Westar. There were no signs of host cell damage until 7 to 8 days postinoculation (dpi), when trypan-blue-stained leaf mesophyll cells were first detected. Hyphae were visible in the intercellular spaces of the inoculated area from 5 dpi and were associated with trypan-blue-stained cells at 8 to 9 dpi. Hallmarks of apoptosis, observed coincident with or immediately prior to the formation of leaf lesions at 8 to 10 dpi, included membrane shrinkage of the mesophyll cell cytoplasm, loss of cell to cell contact in mesophyll cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of nuclei in apparently "healthy" tissue immediately adjacent to dead areas. Hyphae were highly branched and prolific in the "healthy" tissue immediately adjacent to dead areas 9 to 10 dpi, and formed pycnidia inside dead areas 11 to 12 dpi. Coinfiltration of the tetrapeptide caspase inhibitor Ac-DEVD-CHO with spores of the pathogen significantly suppressed development of leaf lesions but did not affect fungus viability. We hypothesize that L. maculans elicits apoptosis as a dependent component of pathogenesis in susceptible B. napus, and that the fungus uses apoptotic cells as a source of nutrition for reproduction and further growth.

Fast moves in arbuscular mycorrhizal symbiotic signalling.

Exciting research looking at early events in arbuscular mycorrhizal symbioses has shown how the fungus and plant get together. Kohki Akiyama et al. have demonstrated that strigolactones in root exudates are fungal germ tube branching factors, and Arnaud Besserer et al. found that these compounds rapidly induce fungal mitochondrial activity. Andrea Genre et al. have shown that subsequent development of appressoria on host roots induces construction of a transient prepenetration apparatus inside epidermal cells that is reminiscent of nodulation infection.

A Novel Tomato Fusarium Wilt Tolerance Gene.

The reduced mycorrhizal colonization (rmc) tomato mutant is unable to form mycorrhiza and is more susceptible to Fusarium wilt compared with its wild-type isogenic line 76R. The rmc mutant has a chromosomal deletion affecting five genes, one of which is similar to CYCLOPS. Loss of this gene is responsible for non-mycorrhizality in rmc but not enhanced Fusarium wilt susceptibility. Here, we describe assessment of a second gene in the rmc deletion, designated Solyc08g075770 that is expressed in roots. Sequence analyses show that Solyc08g075770 encodes a small transmembrane protein with putative phosphorylation and glycosylation sites. It is predicted to be localized in the plasma membrane and may function in transmembrane ion transport and/or as a cell surface receptor. Complementation and knock-out strategies were used to test its function. Some putative CRISPR/Cas-9 knock-out transgenic events exhibited Fusarium wilt susceptibility like rmc and some putative complementation lines were 76R-like, suggesting that the tomato Solyc08g075770 functions in Fusarium wilt tolerance. This is the first study to demonstrate that Solyc08g075770 is the contributor to the Tfw locus, conferring tolerance to Fusarium wilt in 76R which was lost in rmc.

A Lycopersicon esculentum phosphate transporter (LePT1) involved in phosphorus uptake from a vesicular-arbuscular mycorrhizal fungus.

In vesicular-arbuscular mycorrhizal symbioses, specialized fungal structures (the arbuscules) are formed which are in intimate contact with plant root cortical cells. It is assumed that these arbuscules are the major sites of solute transfer between the plant and fungus, but there have been no studies that definitively show the extent or types of transfer processes that occur in this structure. Phosphate is one of the major nutrients that is acquired by mycorrhizal fungi and transferred to plants. In this study a single Lycopersicon esculentum cDNA was cloned and shown to be identical to LePT1, a previously cloned inorganic-phosphate transporter. Expression studies revealed that LePT1 transcript levels remained constant in mycorrhizal plants, but increased in phosphate-starved, non-mycorrhizal plants. Localization of the LePT1 transcript by in situ hybridization showed that this gene is highly expressed in arbuscule-containing cortical cells in mycorrhizal plants. In non-mycorrhizal plants LePT1 expression was localized to the stele and cortex. The expression studies suggest that this transporter is involved in phosphate nutrition of L. esculentum and its localization in cells that contain arbuscules indicate that it may be the mechanism used by the plant to take up phosphate that is effluxed across the fungal plasma membrane of the arbuscule. Based on our findings and those of others, an integrated model of inorganic phosphate uptake and transfer in mycorrhizal and non-mycorrhizal plants is presented.

Position of the reduced mycorrhizal colonisation (Rmc) locus on the tomato genome map.

Our research aims to investigate the molecular communication between land plants and arbuscular mycorrhizal (AM) fungi in the establishment of symbiosis. We have identified a mutation in the facultative AM host tomato, which we named rmc. Plants that are homozygous for rmc no longer host most AM fungi. The mutation also affects the interaction of tomato with root knot nematode and Fusarium wilt. However, the function/s encoded by the intact Rmc locus is/are unknown. To clone and sequence the gene or genes that comprise the Rmc locus, we have initiated a positional cloning project. In this paper, we report the construction of mapping populations and use of molecular markers from the published genome map to identify the location of Rmc on tomato chromosome 8. Nucleotide binding site-leucine rich repeat resistance genes, reported to reside in the same region of that chromosome, provided insufficient differences to develop cleaved amplified polymorphic sequence markers. Therefore, we were unable to map these sequences in relation to rmc. Our results potentiate future work to identify the Rmc function and to determine the genetic basis for the multiple plant-microbe interaction functions that the rmc mutation has defined.

The first gene-based map of Lupinus angustifolius L.-location of domestication genes and conserved synteny with Medicago truncatula.

We report the first gene-based linkage map of Lupinus angustifolius (narrow-leafed lupin) and its comparison to the partially sequenced genome of Medicago truncatula. The map comprises 382 loci in 20 major linkage groups, two triplets, three pairs and 11 unlinked loci and is 1,846 cM in length. The map was generated from the segregation of 163 RFLP markers, 135 gene-based PCR markers, 75 AFLP and 4 AFLP-derived SCAR markers in a mapping population of 93 recombinant inbred lines, derived from a cross between domesticated and wild-type parents. This enabled the mapping of five major genes controlling key domestication traits in L. angustifolius. Using marker sequence data, the L. angustifolius genetic map was compared to the partially completed M. truncatula genome sequence. We found evidence of conserved synteny in some regions of the genome despite the wide evolutionary distance between these legume species. We also found new evidence of widespread duplication within the L. angustifolius genome.

Differential expression of Glomus intraradices genes in external mycelium and mycorrhizal roots of tomato and barley.

Relative quantitative RT-PCR and western blotting were used to investigate the expression of three genes with potentially regulatory functions from the arbuscular mycorrhizal fungus Glomus intraradices in symbiosis with tomato and barley. Standardisation of total RNA per sample and determination of different ratios of plant and fungal RNA in roots as colonisation proceeded were achieved by relative quantitative RT-PCR using universal (NS1/NS21) and organism-specific rRNA primers. In addition, generic primers were designed for amplification of plant or fungal beta-tubulin genes and for plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes as these have been suggested as useful controls in symbiotic systems. The fungal genes Ginmyc1 and Ginhb1 were expressed only in the external mycelium and not in colonised roots at both mRNA and protein levels, with the proteins detected almost exclusively in the insoluble fractions. In contrast, mRNA of Ginmyc2 was identified in both external and intraradical mycelium. In mycorrhizal roots, Ginmyc2 and fungal beta-tubulin mRNAs increased in proportion to fungal rRNA as colonisation proceeded, suggesting that accumulation reflected intraradical fungal growth. Fungal alpha-tubulin protein and beta-tubulin mRNA both appeared to be more abundantly accumulated in AM hyphae within heavily colonised roots than in external hyphae, relative to fungal rRNA. Tomato GAPDH mRNA accumulation was proportional to tomato rRNA, but accumulation of tomato beta-tubulin mRNA was reduced in colonised roots compared to non-mycorrhizal roots. These results provide novel evidence of differential spatial and temporal regulation of AM fungal genes, indicate that the expression of tubulin genes of both plant and fungus may be regulated during colonisation and validate the use of multiple 'control' genes in analysis of mycorrhizal gene expression.