pMVR-hp binary vector (Reddy and Rajam, 2016) , resulting in pMVR-hp-CgCOM1-S. Thus out according to the protocols describe by Mahto et al. (2018) and Madhulatha et al. (2007) , The total genomic DNA was isolated from leaf tissue of putative CgCOM1-RNAi chilli and 1 5 0 tomato lines using CTAB method (Doyle and Doyle, 1990) and employed for molecular Transgenic plants transformed with pMVR-hp-CgCOM1i construct and empty pMVR- Table S1 ). Table 1 ). Subsequently, stem-loop 1 8 3 RT-PCR was performed using siRNA specific forward (see above) and universal reverse 1 8 4 primers to amplify 60 bp amplicon encompassing siRNA from transgenic leaves. Low 1 8 5 molecular weight RNA isolated from wild-type plant served as a control. RNAi (Pusa Rohini-CgCOM1i and Pusa Early Dwarf-CgCOM1i) lines using fungal spore 1 9 1 inoculum (1×10 5 spores ml -1 ), prepared from 3-days old cultures as described above. Empty 1 9 2 vector transformed (EV) and wild type (WT) plants challenged with the fungal pathogen 1 9 3 served as controls. Inoculation in the case of leaves was performed on abaxial surface. And in 1 9 4 the case of fruits, surface of the fruit was first pricked with a toothpick and then inoculum 1 9 5 was applied. Infected leaves and fruits were incubated at 25 o C inside a humidified plastic 1 9 6 box, and allowed for the development of the disease and progression of lesion formation. For 1 9 7 assessing the progression of disease symptoms, leaves and fruits were photographed at 1 9 8 regular intervals (0 -10 days post inoculation) and used for measuring infected area with 1 9 9
Image-J analysis program (https://imagej.nih.gov/ij). Experiments were repeated three times 2 0 0 with 5 biological samples each. Percentage of infected area in leaf and fruit was calculated as 2 0 1 the infected leaf or fruit area/total leaf or fruit area X 100. The t-test was performed on 2 0 2 calculated infected leaf or fruit areas (%) for determining significance (p≤0.0001). intervals (at 0, 7, 36, 120 hpi and 9 dpi) and used for determining CgCOM1 expression levels. For these studies total RNA was isolated from the infected tissues, cDNA prepared and used 2 0 7 for assessing CgCOM1 transcript abundance by semi-quantitative RT-PCR and quantitative 2 0 8 RT-qPCR using appropriate primer sets (Supplementary Table 1 In ascomycetous Magnaporthe oryzae, insertional inactivation as well as deletion of COM1 2 2 6 (MoCOM1) gene produced defects in conidial development, appressoria formation, MoCOM1 gene encodes for a helix-loop-helix type transcription regulator, which is 2 3 0 unique to filamentous ascomycetes ( Supplementary Fig. S2 ). CgCOM1 (accession no. (Supplementary Fig. S3 ). From among the species analyzed, COM1 proteins from C. Glomerellaceae showed maximum resemblance with highly conserved protein sequences (76- infection processes of M. oryzae (see above), we hypothesized that COM1 gene in C. gloeosporioides also likely plays a similar role in contributing to infective capability and anthracnose disease. So in current study we aimed to interrupt the development of C. to plant genes to avoid off-target effects. The selected CgCOM1 gene fragment was amplified 2 5 9
using gene specific primers (Supplementary Table S1 ) and cloned into pBlueScript SK. For Table S2 ). 
In order to assess resistance of transgenic plants against anthracnose disease, challenge assays 2 8 8
were performed with C. gloeosporioides against detached leaves and fruits derived from the 2 8 9
CgCOM1-RNAi chilli (PJ-CgCOM1i-1, -3, -9 and PSB-CgCOM1i-1, -4, -6) and tomato (PR-
CgCOM1i-1, -2 and PED-CgCOM1i-1, -5) transgenic lines as described in materials and impeded growth of the fungus and spread of disease, as compared to wild type and vector 2 9 7
control plants (Fig. 2) . In wild type and vector control chilli and tomato leaves, water soaked 2 9 8 spots with necrotic lesions, typical of anthracnose disease symptoms caused by C. and 6, lesion areas in leaves attained only a maximum of 2.6%, 1.3% and 2%, respectively, as In tomato, at 3DPI, infected area on the leaves of the PR-CgCOM1i transgenic lines 1 3 1 3
and 2 averaged around 3%, whereas in the PED-CgCOM1i transgenic lines 1 and 5 it was 3 1 4 about 3.4%, in comparison to 11.2% and 11.5% in the vector control plants PR-EV and PED-3 1 5 EV, respectively (Fig. 2G, H) . Progression of disease was slower in the transgenic lines of 3 showing the spread of the disease) in leaves of CgCOM1-RNAi chilli and tomato lines was 3 2 3 about 65-93% lesser than in the WT and EV plants at 10 DPI and 7 DPI respectively (Fig. 2) . comparison to wild type and vector control plants (Fig.3) . Water soaked necrotic lesions diminished lesion formation in comparison to EV and WT fruits (Fig. 3) . Percentage of 78.28% in PSB-CgCOM1i-1 compared to EV challenged plants (Fig. 3E, F) .
The percentage of infected area in tomato fruits of PR-CgCOM1i-1 transgenic lines was infected area was 7.07% in PED-CgCOM1i-1 and 82.02% in vector control fruits (Fig. 3H ).
Disease progression in wild type and vector control fruits was almost similar. To validate the inhibition of disease progression is due to silencing of the CgCOM1 gene, COM1 homologue as a potential target for host induced gene silencing in C. gloeosporioides.
In order to neutralize the growth of the fungal pathogen C. gloeosporioides by HIGS, we intact COM1 transcripts albeit at low levels (before they were being completely degraded due tubes and appressoria adhered to the surface of the inoculated leaves (Fig. 6) . In our studies ( Fig. 6) . Similar type of infection was also observed in guava (Moraes et al., 2013) . However, 4 4 9
in cowpea leaves, it was reported that C. gloeosporioides attempt to gain entry into leaf In our study, RT-PCR and RT-qPCR analyses indicated that the expression of Our studies demonstrated that HIGS is a powerful tool to control anthracnose disease Table S1 . Primers used in the present study. infected and uninfected wild type chilli fruits by RT-qPCR.
Acknowledgements 5 0 4
The research work was financed by the Department of Biotechnology (Grant No. Government of India, for providing fellowship. 