Construction of the AcTPR2-TRV2 vector
A silencing fragment was cloned and its sequence was the same as that of the reference AcTPR2 (Ach25g228601.2-TA, http://kiwifruitgenome.org/). An AcTPR2-TRV2 construct was generated by introducing a 446-bp XbaI/BamHI DNA fragment into a pTRV2 vector (Fig. 1A and 1B). PCR detection of resistant colonies bearing the AcTPR2-TRV2 construct was performed to confirm that the silencing fragment was successfully ligated onto the pTRV2 vector (Fig. 1C).
AcTPR2 expression was greatly reduced in AcTPR2-TRV fruits
Agrobacterium GV3101 harboring the AcTPR2-TRV2 expression vector (AcTPR2-TRV), sterilized ddH2O (WT), and the vector pTRV1-2 (TRV), were transformed to 'Hongyang' kiwifruit by transient injection. AcTPR2 expression was measured for all three groups. AcTPR2 level was markedly downregulated in the AcTPR2-TRV fruits at 6 days post-injection. In contrast, there were no drastic differences in AcTPR2 expression between the WT and TRV groups (Fig. 2).
Virus-induced silencing of AcTPR2 enhanced kiwifruit susceptibility to B. cinerea
AcTPR2 expression was compared between AcTPR2-TRV and control kiwifruit after B. cinerea infection. The AcTPR2 level was highest at 4 days post-inoculation (dpi) and the infection time was prolonged in the WT, TRV empty vector, and AcTPR2-TRV fruits. The AcTPR2 levels in these treatments were nearly sixfold higher than they were in the control at 1 dpi. However, the AcTPR2 level in AcTPR2-TRV was twofold lower than it was at 1 dpi (Fig. 3).
The lesion areas on AcTPR2-silenced kiwifruit were larger than those for the control groups. Furthermore, the injection sites were more susceptible to rot in the AcTPR2-silenced kiwifruit than they were in the control. At 5 dpi, the lesion areas on the AcTPR2-TRV fruit were nearly threefold larger than they were on the WT and TRV vector fruits. The B. cinerea load was also significantly higher in the AcTPR2-TRV fruits than that in the control at 4 dpi and 5 dpi. Thus, virus-induced silencing of AcTPR2 enhanced kiwifruit susceptibility to B. cinerea (Fig. 4).
Kiwifruit activate pathogen resistance-related defense enzymes in response to infection
The activity levels of enzymes such as SOD, POD, CAT, and PAL are indicative of plant disease resistance. These enzymes may be upregulated in response to biotic and abiotic stress and enhance host resistance. The activity levels of all four defense enzymes increased in the control and AcTPR2-TRV kiwifruits in response to B. cinerea infection. SOD, POD, and PAL rapidly reacted to pathogenesis at 1 dpi and their activity levels continued to rise until 4 dpi, but decreased by 5 dpi. CAT was first induced at 2 dpi and its activity steadily increased with infection time. However, at 5 dpi, its activity declined. In the absence of B. cinerea infection, the activity levels of all four enzymes were much higher in AcTPR2-TRV than they were in the control. Nevertheless, the enzyme activity markedly increased in the AcTPR2-TRV groups under infection stress (Fig. 5).
Increased B. cinerea susceptibility in the AcTPR2-TRV groups is related to phytohormone interactions
TPL/TPR proteins participate in plant signaling pathways and the various phytohormones interact. Here, the relative levels of the phytohormones, IAA, GA3, ABA, and SA, were measured. IAA, GA, and SA levels were observed to sharply increase before 4 dpi, and thereafter, they decreased rapidly. In the absence of B. cinerea infection, IAA, GA, and SA levels were substantially higher in AcTPR2-TRV than those in the control WT and TRV groups. Upon infection, the levels of IAA, GA, and SA were higher in AcTPR2-TRV than they were in the uninfected AcTPR2-TRV at 1–4 dpi. However, the levels of these phytohormones declined at 5 dpi (Fig. 6A, 6B, and 6D). In the control, the ABA content continuously increased as the Botrytis cinerea infection prolonged. In case of the AcTPR2-TRV treatment at 1–2 dpi, the ABA content was higher following B. cinerea infection than that in the uninfected fruits. After 2 dpi, however, the ABA level considerably fell in the AcTPR2-TRV kiwifruit (Fig. 6C).
Virus-induced silencing of AcTPR2-induced IAA signaling gene expression
Here, we used qRT-PCR to measure the expression levels of the genes governing auxin biosynthesis and signaling in the AcTPR2-TRV and control fruits. AcNIT1 was slightly upregulated in the control fruit within 5 days of storage, but markedly increased upon B. cinerea infection, especially at 1–3 dpi. The AcNIT1 level was higher in the AcTPR2-TRV than that in the control fruits (Fig. 7A). AcARF1and AcARF2 were somewhat upregulated in the control but their levels were fourfold higher in the AcTPR2 silenced fruits than those in the control. AcARF1and AcARF2 were strongly induced in response to B. cinerea infection (Fig. 7B and 7C).