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Microorganisms
2024 Mar 28;124:. doi: 10.3390/microorganisms12040681.
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Influence of Two Hexose Transporters on Substrate Affinity and Pathogenicity in Magnaporthe oryzae.
Huang T
,
Guo D
,
Luo X
,
Chen R
,
Wang W
,
Xu H
,
Chen S
,
Lin F
.
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Hexose transporters (HXT) play a crucial role in the pathogenicity of Magnaporthe oryzae, serving not only as key facilitators for acquiring and transporting sugar nutrients to support pathogen development, but also as sugar sensors which receive transduction signals. The objective of this study is to investigate the impact of MoHXT1-3 on rice pathogenicity and hexose affinity. MoHXT1-3 deletion mutants were generated using CRISPR/Cas9 technology, and their affinity for hexose was evaluated through yeast complementation assays and electrophysiological experiments in Xenopus oocytes. The results suggest that MoHXT1 does not contribute to melanin formation or hexose transportation processes. Conversely, MoHXT2, despite displaying lower affinity towards the hexoses tested in comparison to MoHXT3, is likely to have a more substantial impact on pathogenicity. The analysis of the transcription profiles demonstrated that the deletion of MoHXT2 caused a decrease in the expression of MoHXT3, whereas the knockout of MoHXT3 resulted in an upregulation of MoHXT2 transcription. It is noteworthy that the MoHXT2M145K variant displayed an incapacity to transport hexoses. This investigation into the functional differences in hexose transporters in Magnaporthe oryzae provides insights into potential advances in new strategies to target hexose transporters to combat rice blast by blocking carbon nutrient supply.
No. 2014A050503056 the Project of Science and Technology in Guangdong province, ZhiZhong2022-01 Guangdong Provincial Key Laboratory of High Technology for Plant Protection
Figure 1. Changes to pathogenicity-associated phenotypic in ΔMohxt2 and ΔMohxt3 strains compared with the wild type A60. (A) Growth of the mutants ΔMohxt2 and ΔMohxt3. Strains were cultivated on CM supplemented with various carbon sources, including glucose, mannose, galactose or fructose. A parallel control lacking any carbon source (CM-NC) was included. (B) Assessment of conidiation for the stains on CM plates by preparation of conidial suspension. Means are expressed as the number of conidia ×106 mL−1 of the conidial suspension (cm−2) of the culture. Error bars represent standard deviations. Independent sample t-tests were used to determine significant differences in count data, and “*” represents significant differences between strains (p < 0.05). (C) Conidial germination ability of the wild-type and mutant strains. Drops of conidial suspension were placed on hydrophobic coverslips and kept in a moist chamber at 25 °C for 0, 12, 24 and 36 h before imaging under a light microscope. Scale bar = 100 μm. (D) Calculation of disease spot number produced by the strains. Independent sample t-tests were used to determine significant differences in count data and “*” represent significant differences between strains (p < 0.05). (E) Infection assays of strains on rice leaves. Droplets of conidial suspensions (1 × 105 spores mL−1) were inoculated on 15-day-old rice seedlings (n ≥ 10) (CO39). Photographs were taken at 96 hpi. Red triangles indicate symptoms of onset. (F) Observation of mycelium morphological features using scanning electron microscopy (SEM). Scale bars are 5 μM for A60, ΔMohxt2 and ΔMohxt3.
Figure 2. Hexose transport by MoHXT1-3 in Xenopus laevis oocytes. (A) Inward currents induced by hexoses in X. laevis oocytes expressing MoHXTs. (B–E) Kinetic analysis of glucose, fructose and mannose transport mediated by MoHXT2 and MoHXT3 in X. laevis oocytes, where (B,C) show the fitted curve for MoHXT2 and (D,E) show the fitted curve for MoHXT3. X. oocytes expressing the two MoHXTs were kept in Kulori solution at pH 5.0 with a clamping potential of −40 mV. Currents were normalized to Vmax. (D,E) Current–voltage relationship for MoHXT2 and MoHXT3 channels induced by glucose, fructose and mannose, respectively. The oocytes were clamped at −40 mV and stepped down in test voltage from 40 to −140 mV over 300 ms in −20 mV increments. The substrate-induced current (background subtraction) was measured at −140 mV. Error bars represent the mean ± SE (n = 3).
Figure 3. Hexose transport by MoHXT2 and MoHXT3 in EBY.VW4000 yeast cells. (A) Schematic diagrams of relative positions for each yeast strain and the media they were grown on. (B) EBY.VW4000 expression of MoHXT2 and MoHXT3 grown on a medium containing hexose. The growth of EBY.VW4000 carrying the vector pDR195 was set up as a negative control in each plate, while a medium containing 2% maltose (disaccharide) was used as a positive control. Complementary yeast strains carrying pDR195-Mohxt2 and pDR195-Mohxt3 were spotted on media in addition to the indicated monosaccharide (glucose, mannose or fructose) and incubated at 30 °C for 144 h. These experiments were repeated three times (with three different transformants, providing three biological replicates), and similar results were obtained in each case.
Figure 4. Hexose transport ability of MoHXT2 variants. (A) Growth of yeast strains expressing MoHXT2, MoHXT2M145K, MoHXT2A89R and MoHXT2K421A on media containing 2% glucose, mannose or fructose, respectively. A positive control was employed, consisting of a medium supplemented with maltose. The yeast cell cultures were diluted in a five-fold gradient, subsequently cultivated in an incubator at a temperature of 30 °C for a duration of 3 days and then photographed. (B) Current–voltage reduced by different substrates in Xenopus oocytes expressing MoHXT2M145K. Oocytes were maintained in a Kulori media solution at pH 5.0 at a perfusion current of −40 mV and depolarized with Vmax. (C) The oocytes were clamped at −40 mV and stepped down to a test voltage between 40 and −140 mV over 300 ms in −20 mV increments. The substrate-induced current (background subtraction) was measured at −140 mV. Error bars represent the mean ± SE (n = 3).
Figure 5. Transcription profiles of the hexose transporter-like genes in A60 and the ΔMohxt2 and ΔMohxt3 strains. (A) The fragments per kilobase of exon per million mapped fragments (FPKM) values for each gene are shown in Table S4. MGG_08617 and MGG_10293, which are included in the phylogenetic tree in Figure S1, are excluded from the heatmap as they were not detected in the RNA-Seq experiment. The genomes were classified based on the comparability of their expression pattern, showing the differences compared to A60. (B) Venn diagram of changes in genes expression levels among the three strains.
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