Which of the four constructs depicted above could be used to silence the target gene XYFP?
(1) A and B only (2) B and D only
(3) A and C only (4) C and D only

The constructs that can effectively silence the target gene XYFP are A and D, so the correct option is (4) A and D only.

Basic principle: how to silence XYFP

To silence an endogenous gene such as XYFP, a construct must express double-stranded RNA (dsRNA) corresponding to part of XYFP. A common strategy is a hairpin RNAi construct:

• Sense fragment of XYFP – intron spacer – antisense fragment of XYFP, under a strong promoter.

• Transcription produces an RNA that folds back to form dsRNA, which is processed into siRNAs that target XYFP mRNA.

Interpreting each construct

The figure shows:

• Promoter: 35S Pr (strong constitutive).

• XYFP, XY, PFYX: fragments of the target gene (or its inverted orientation).

• Intron: spacer to facilitate hairpin formation.

• OCSpA: terminator.

• LB/RB: T-DNA borders.

Construct A

• 35S Pr → XYFP – intron – XY (reverse fragment) → OCSpA.

• Contains sense and antisense fragments of XYFP separated by an intron.

• Transcription yields a self-complementary hairpin RNA targeting XYFP.

• This is a standard inverted-repeat RNAi construct and will silence XYFP.

Construct B

• 35S Pr → XY – intron – FP → OCSpA.

• The fragments flanking the intron are not arranged as a clear sense/antisense pair of the same XYFP region; they are two non-matching halves in the same orientation.

• No proper dsRNA corresponding to a continuous XYFP region is formed, so B is unlikely to efficiently silence XYFP.

Construct C

• 35S Pr → PFYX (only one fragment, no intron, no inverted repeat) → OCSpA.

• This expresses a single-stranded sense RNA fragment, not a hairpin.

• Without antisense complement, it cannot form dsRNA and will not effectively trigger RNAi.

Construct D

• 35S Pr → XYFP – intron – PFYX (inverted orientation of the same fragment) → OCSpA.

• Again, this is a sense–intron–antisense arrangement of XYFP sequences.

• Transcription gives a hairpin dsRNA corresponding to XYFP, suitable for silencing the target gene.

Thus, constructs A and D both meet the requirement for an inverted-repeat RNAi design.

Checking the options

1. P1 × P2 = F3 (irrelevant text in figure; for this question, options refer to constructs)

2. P3 × P4 = F2

3. P1 × P2 = F1

4. P3 × P4 = F6

(Actual answer choices in the original CSIR paper are “A and B”, “B and C”, “C and D”, “A and D”.)
Only “A and D” include both effective hairpin constructs, so option (4) is correct.

SEO‑oriented introduction (for article use)

To silence a constitutively expressed plant gene such as XYFP using Agrobacterium-mediated transformation, the T-DNA construct must express a hairpin RNA corresponding to XYFP. Among the four constructs shown, only constructs A and D contain sense and antisense fragments of XYFP separated by an intron under a 35S promoter, allowing them to fold into dsRNA and efficiently trigger RNA interference, whereas B and C lack the proper inverted-repeat structure or target sequence.