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Heterologous expression of key components of the Magnaporthe grisea ACE1 gene cluster produces a potential precursor of cryptic avirulence signalling compounds that induce resistance to M. grisea in rice., The ACE1 and RAP1 genes from the avirulence signalling gene cluster of the rice blast fungus Magnaporthe oryzae were expressed in Aspergillus oryzae and M. oryzae itself. Expression of ACE1 alone produced a polyenyl pyrone (magnaporthepyrone), which is regioselectively epoxidised and hydrolysed to give different diols, 6 and 7, in the two host organisms. Analysis of the three introns present in ACE1 determined that A. oryzae does not process intron 2 correctly, while M. oryzae processes all introns correctly in both appressoria and mycelia. Co-expression of ACE1 and RAP1 in A. oryzae produced an amide 8 which is similar to the PKS-NRPS derived backbone of the cytochalasans. Biological testing on rice leaves showed that neither the diols 6 and 7, nor amide 8 was responsible for the observed ACE1 mediated avirulence, however, gene cluster analysis suggests that the true avirulence signalling compound may be a tyrosine-derived cytochalasan compound.

Heterologous expression of genes from the proposed aspyridone biosynthetic cluster from Aspergillus nidulans in the host Aspergillus oryzae led to the production of eight different compounds in addition to aspyridone A 1, one of the previously observed products. The pathway genes were incapable of producing aspyridone B 2, the previously accepted final product of the pathway. Expression of restricted sets of genes in addition to the core polyketide synthase – nonribosomal peptide synthetase (PKS-NRPS) genes apdA and apdC revealed: that apdE encodes a cytochrome P450 enzyme with ring-expanding and unprecedented dephenylation activity; that apdB encodes an N-hydroxylase, an activity not previously suspected; that the productivity of ApdA and ApdC proteins appears to be significantly enhanced in the presence of the downstream ApdE oxidase; and no obvious chemical roles for ApdD and ApdG. Furthermore, the ApdC enoyl reductase appears to operate with different stereoselectivity in different PKS cycles. All of these features illustrate the inherent diversity of compounds potentially produced by the apd pathway and the high utility of a whole pathway expression strategy for investigating and revealing new biosynthetic chemistry in fungi. Government of Pakistan Majlis Amanah Rakyat (MARA) Malaysia EP/F066104/1

The plant hormone auxin controls root epidermal cell development in a concentration-dependent manner. Root hairs are produced on a subset of epidermal cells as they increase in distance from the root tip. Auxin is required for their initiation and continued growth, but little is known about its distribution in this region of the root. Contrary to the expectation that hair cells might require active auxin influx to ensure auxin supply, we did not detect the auxin-influx transporter AUX1 in root-hair cells. A high level of AUX1 expression was detected in adjacent non-hair cell files. Non-hair cells were necessary to achieve wild-type root-hair length, although an auxin response was not required in these cells. Three-dimensional modelling of auxin flow in the root tip suggests that AUX1-dependent transport through non-hair cells maintains an auxin supply to developing hair cells as they increase in distance from the root tip, and sustains root-hair outgrowth. Experimental data support the hypothesis that instead of moving uniformly though the epidermal cell layer, auxin is mainly transported through canals that extend longitudinally into the tissue.

To explore the role of auxin-binding protein (ABP1) in planta, a number of transgenic tobacco (Nicotiana tabacum) lines were generated. The wild-type KDEL endoplasmic reticulum targeting signal was mutated to HDEL, another common retention sequence in plants, and to KEQL or KDELGL to compromise its activity. The auxin-binding kinetics of these forms of ABP1 were found to be similar to those of ABP1 purified from maize (Zea mays). To test for a physiological response mediated by auxin, intact guard cells of the transgenic plants were impaled with double-barreled microelectrodes, and auxin-dependent changes in K(+) currents were recorded under voltage clamp. Exogenous auxin affected inwardly and outwardly rectifying K(+) currents in a dose-dependent manner. Auxin sensitivity was markedly enhanced in all plants overexpressing ABP1, irrespective of the form present. Immunogold electron microscopy was used to investigate the localization of ABP1 in the transgenic plants. All forms were detected in the endoplasmic reticulum and the KEQL and KDELGL forms passed further across the Golgi stacks than KDEL and HDEL forms. However, neither electron microscopy nor silver-enhanced immunogold epipolarization microscopy revealed differences in cell surface ABP1 abundance for any of the plants, including control plants, which indicated that overexpression of ABP1 alone was sufficient to confer increased sensitivity to added auxin. Jones et al. ([1998] Science 282: 1114-1117) found increased cell expansion in transgenic plants overexpressing wild-type ABP1. Single cell recordings extend this observation, with the demonstration that the auxin sensitivity of guard cell K(+) currents is mediated, at least in part, by ABP1.

Arachidonic acid (C20:4 Δ5,8,11,14)) is a polyunsaturated fatty acid synthesized by the ?-fatty acid desaturation of di-homo-?-linolenic acid (C20:3 Δ8,11,14)). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungus Mortierella alpina. We have isolated a cDNA encoding the Δ5-fatty acid desaturase from M. alpina via a polymerase chain reaction- based strategy using primers designed to the conserved histidine box regions of microsomaL desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic acid. The M. alpina Δ5- desaturase is the first example of a cloned Δ5-desaturase, and differs from other fungal desaturases previously characterized by the presence of an N- terminal domain related to cytochrome b5.