تغییر بیان ژن‌های مرتبط با اتوفاژی در پاسخ به سرکوبگر خاموشی HC-Pro ویروس ای سیب‌ زمینی (Potato virus A) در گیاه Nicotiana benthamiana

نوع مقاله : مقاله کامل پژوهشی

نویسندگان

1 دانشجویان دکتری، مرکز تحقیقات ویروس شناسی گیاهی، دانشکده کشاورزی، دانشگاه شیراز.

2 دانشیار مرکز تحقیقات ویروس شناسی گیاهی، دانشکده کشاورزی، دانشگاه شیراز

3 استاد مرکز تحقیقات ویروس شناسی گیاهی، دانشکده کشاورزی، دانشگاه شیراز.

چکیده

در سال­های اخیر نقش اتوفاژی در خاموشی آر­ان­ای القاء شده توسط ویروس از طریق فعالیت پروتئین­های سرکوبگر خاموشی آر­ان­ای به اثبات رسیده است. به منظور درک بهتر نقش احتمالی سرکوبگر خاموشی HC-Pro ویروس ای سیب ­­زمینی در فرآیند اتوفاژی (autophagy)، اثر این سرکوبگر ویروسی روی بیان تعدادی از ژن­های مهم و دخیل در اتوفاژی مورد بررسی قرار گرفت. ژنHC-Pro  ویروس ای سیب ­­زمینی در ناقل بیان pGWB17 با برچسبmyc  در انتهای کربوکسیلی و پروموتور 35S در انتهای آمینی توسط تکنولوژی  gatewayهمسانه­سازی شد. کشت­های اگروباکتریوم حاوی  HC-Proویروس ای سیب ­زمینی با استفاده از سرنگ در ناحیه پشتی برگ­های گیاه Nicotiana benthamianaتزریق شدند. بیان ژن­های ATG2، ATG7،  ATG6و AGO1در پاسخ به سرکوبگر خاموشی HC-Pro ویروس ای سیب ­زمینی، در فاصله زمانی پنج روز پس از تزریق، با استفاده از تکنیک ریل تایم پی­سی­آر (Real-Time PCR) اندازه­گیری شدند. سرکوبگر خاموشیHC-Pro   ویروس ای سیب ­زمینی میزان بیان ژن­های ATG6، ATG2و ATG7را به میزان ۸۹/۵، ۳/۷ و ۶/۷ برابر نسبت به برگ­های تزریق شده با سازه حاوی پلاسمید خالی pGWB افزایش داد. در حالی­که میزان رونوشت  AGO1به­عنوان جزء کلیدی کمپلکس خاموشی القاء شده توسط آر­ان­ای، به میزان ۳۶/۱ برابر نسبت به میزان آن در برگ­های فاقد  HC-Proکاهش نشان داد. نتایج این تحقیق حاکی از دخالت ژن­های ATG2، ATG7، ATG6و AGO1در پاسخ­های دفاعی گیاه  N .benthamianaدر مقابل سرکوبگر خاموشی HC-Pro ویروس ای سیب­ زمینی و امکان استفاده از آن در تدوین روش­های جدید مدیریت ویروس­های گیاهی می­باشد.

کلیدواژه‌ها


عنوان مقاله [English]

Altered expression of autophagy-related genes in Nicotiana benthamiana plants in response to Potato virus A HC-Pro silencing suppressor

نویسندگان [English]

  • A. Tahmasebi 1
  • A. Afsharifar 2
  • S. Rabiee 1
  • K. Izadpanah 3
1 . Ph.D. student of Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran.
3 Professor, Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran.
چکیده [English]

Accumulating data have revealed the role of autophagy in virus-induced RNA silencing via viral RNA silencing suppressor activity. To extend our understanding for the possible role of Potato virus A-HC-Pro (PVA-HC-Pro) suppressor protein in autophagy process, we investigated the effect of PVA-HC-Pro on expression of some important genes involved in autophagy. The cDNA of ORF PVA HC-Pro was cloned in pGWB17 vector with a C-terminal myc tag and N-terminal 35S promoter using Gateway technology. Agrobacterium cultures harboring PVA-HC-Pro were infiltrated into the abaxial side of N. benthamiana leaves. The expression of ATG6, ATG2, ATG7 and AGO1 genes were measured at 5 days post infiltration in response to PVA-HC-Pro using qRT-PCR technique. PVA HC-Pro as a suppressor of RNA silencing increased the expression level of ATG6, ATG2 and ATG7 5.89, 7.3 and 7.6 fold, respectively, compared to corresponding values in leaves infiltrated with empty plasmid (EP) as a control. In contrast, the transcript level of Argonaute1 (AGO1), a key component of RNA-induced silencing complex (RISC), was decreased by 1.36-fold compared to the level of AGO1in infiltrated leaves without PVA-HC-Pro. Results of this study indicated that PVA HC-Pro can alter the expression of autophagy related genes in N. benthamiana plant. These findings suggest the involvement of ATG6, ATG2, ATG7 and AGO1 in defense responses of N. benthamiana against PVA-HC-Pro, which might be considered in plant breeding programs as a novel approach to the control of plant viruses.

کلیدواژه‌ها [English]

  • : Autophagy
  • HC-Pro
  • N. benthamiana
  • RNA silencing
  • Suppressor of RNA Silencing
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