Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan, 430079, China.
Yunnan Higher Education Institutions, College of Life Science and Technology, Honghe University, Mengzi, 661199, China.
BMC Genomics. 2020 Feb 12;21(1):156. doi: 10.1186/s12864-020-6564-6.
Penicillium italicum (blue mold) is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (< 10 °C) during shipping and storage. P. italicum isolates with considerably high resistance to sterol demethylation inhibitor (DMI) fungicides have emerged; however, mechanism(s) underlying such DMI-resistance remains unclear. In contrast to available elucidation on anti-DMI mechanism for P. digitatum (green mold), how P. italicum DMI-resistance develops has not yet been clarified.
The present study prepared RNA-sequencing (RNA-seq) libraries for two P. italicum strains (highly resistant (Pi-R) versus highly sensitive (Pi-S) to DMI fungicides), with and without prochloraz treatment, to identify prochloraz-responsive genes facilitating DMI-resistance. After 6 h prochloraz-treatment, comparative transcriptome profiling showed more differentially expressed genes (DEGs) in Pi-R than Pi-S. Functional enrichments identified 15 DEGs in the prochloraz-induced Pi-R transcriptome, simultaneously up-regulated in P. italicum resistance. These included ATP-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, ergosterol (ERG) anabolism component genes ERG2, ERG6 and EGR11 (CYP51A), mitogen-activated protein kinase (MAPK) signaling-inducer genes Mkk1 and Hog1, and Ca/calmodulin-dependent kinase (CaMK) signaling-inducer genes CaMK1 and CaMK2. Fragments Per Kilobase per Million mapped reads (FPKM) analysis of Pi-R transcrtiptome showed that prochloraz induced mRNA increase of additional 4 unigenes, including the other two ERG11 isoforms CYP51B and CYP51C and the remaining kinase-encoding genes (i.e., Bck1 and Slt2) required for Slt2-MAPK signaling. The expression patterns of all the 19 prochloraz-responsive genes, obtained in our RNA-seq data sets, have been validated by quantitative real-time PCR (qRT-PCR). These lines of evidence in together draw a general portrait of anti-DMI mechanisms for P. italicum species. Intriguingly, some strategies adopted by the present Pi-R were not observed in the previously documented prochloraz-resistant P. digitatum transcrtiptomes. These included simultaneous induction of all major EGR11 isoforms (CYP51A/B/C), over-expression of ERG2 and ERG6 to modulate ergosterol anabolism, and concurrent mobilization of Slt2-MAPK and CaMK signaling processes to overcome fungicide-induced stresses.
The present findings provided transcriptomic evidence on P. italicum DMI-resistance mechanisms and revealed some diversity in anti-DMI strategies between P. italicum and P. digitatum species, contributing to our knowledge on P. italicum DMI-resistance mechanisms.
意大利青霉（蓝绿霉）是一种柑橘病原体，即使在运输和储存过程中严格控制在低温（<10°C）下，也会导致柑橘果实腐烂。具有相当高抗性的青霉意大利亚种对甾醇脱甲基抑制剂（DMI）杀菌剂的分离株已经出现；然而，这种 DMI 抗性的机制尚不清楚。与已阐明的对青霉绿僵菌（绿僵菌）的抗 DMI 机制相比，青霉意大利亚种如何产生 DMI 抗性尚未得到澄清。
本研究为两种青霉意大利亚种（对 DMI 杀菌剂高度敏感（Pi-S）和高度抗性（Pi-R））制备了 RNA 测序（RNA-seq）文库，以确定在有和没有丙环唑处理的情况下，促进 DMI 抗性的丙环唑反应基因。丙环唑处理 6 小时后，比较转录组分析显示 Pi-R 中的差异表达基因（DEG）比 Pi-S 多。功能丰富性鉴定了丙环唑诱导的 Pi-R 转录组中的 15 个 DEG，同时在青霉意大利亚种抗性中上调。其中包括 ATP 结合盒（ABC）转运蛋白编码基因、主要易化因子超家族（MFS）转运蛋白编码基因、麦角甾醇（ERG）生物合成成分基因 ERG2、ERG6 和 EGR11（CYP51A）、丝裂原激活蛋白激酶（MAPK）信号诱导基因 Mkk1 和 Hog1，以及钙/钙调蛋白依赖性激酶（CaMK）信号诱导基因 CaMK1 和 CaMK2。Pi-R 转录组的片段每百万映射读数的每千碱基（FPKM）分析显示，丙环唑诱导了另外 4 个非编码基因的 mRNA 增加，包括另外两种 ERG11 同工型 CYP51B 和 CYP51C 以及 Slt2-MAPK 信号所需的其余激酶编码基因（即 Bck1 和 Slt2）。我们的 RNA-seq 数据集中获得的所有 19 个丙环唑反应基因的表达模式均通过定量实时 PCR（qRT-PCR）进行了验证。这些证据共同描绘了青霉意大利亚种抗 DMI 机制的总体特征。有趣的是，本研究中 Pi-R 采用的一些策略在先前记录的丙环唑抗性青霉绿僵菌转录组中没有观察到。其中包括所有主要的 EGR11 同工型（CYP51A/B/C）的同时诱导、ERG2 和 ERG6 的过表达以调节麦角甾醇生物合成，以及 Slt2-MAPK 和 CaMK 信号通路的同时动员，以克服杀菌剂诱导的应激。
本研究提供了青霉意大利亚种 DMI 抗性机制的转录组证据，并揭示了青霉意大利亚种和青霉绿僵菌之间抗 DMI 策略的一些差异，为我们了解青霉意大利亚种 DMI 抗性机制提供了帮助。
