Kwansei Gakuin University
Report of Research Outcome
2020/02/04

To President
Department： Science and Technology
Position： Postdoctoral fellow
Name： Vandana Deopa
I report the outcome of the research as follows.
□Sabbatical leave with grantName of theFund/Prog ra m

□KGU Joint Research□Sabbatical leave with no gra nt□Individual Special Research⊠Postdoctora l fell ow※ Please report by desi gnated f orm as f or “Inte rnational Research
Collaboration”.
1.Functional

characte rizationofput ative

chloroplastenvelopeloc atedtransporte rs belonging to the SLC -4 f amily by CR ISPR/Cas9 nickase
ResearchThe me(D10A) mediate d geno me editing in the marine d iatoms, P. tricornutu m a ndT. pseudonana.
2. Study of the Pyshell proteins homologs in T. pseudonana and theirf unctional characterization by C RISPR/Cas9 nickase (D10A) mediat ed
genome editing.

ResearchSite/Venue

Research
periodMatsuda lab, Building I V, Depart men t of Bioscience, Kwansei Gakuin
University, Kobe-Sand a Campus

2019/06/01～

2020/01/31 (8 months ）◆ Summary of the research outcome （ appr ox . 2 ,5 00 wo r ds）
Please write down the outcomes in detail regarding the research theme above.

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Enhancing crop productivity by introduction of carbon concentrating mechanisms
has been a highly sought-after area in the field of photosynthetic research. Amongthe diverse kinds of CCMs present in nature, the diatomic CCM has invoked
greater interest because of its endosymbiotic origin which has enabled it topossess genes from algae, as well as animals and bacteria. Notably, theendosymbiotic event bestowed the diatoms with a four layered chloroplastenvelope i.e. a double-layered chloroplast envelope that, in turn, is encircled by adouble-layered chloroplastic endoplasmic reticulum. Although the information
regarding the function of these envelopes is still scarce, it is well established thatthese play an important role in DIC trafficking. It has been ascertained thatdifferent set of transporters are present on these membranes which along with CAs
in specific compartment function in conjunction to facilitate the uptake of HCO 3 into the chloroplast stroma from the cytoplasm (Figure no.1).

Figure 1. The CO 2 concentrating mechanism in diatoms
Nakajima et al. (2013) identified seven SLC4 genes and three SLC26 genes in thegenome of Phaeodactylum tricornutum. Out of the seven SLC4 genes, PtSLC4-1,PtSLC4-2, and PtSLC4-4 have been already identified as the SLCs located on theplasma membrane which are involved in regulating the influx of HCO 3 - into thecell (Nakajima et al., 2013). The other two, PtSLC4-6 and PtSLC4-7 encode
proteins predicted to localize in the four-layered chloroplast membrane systems as
evident by the presence of targeting signals viz., GSA-FTS and SAA-FHT
respectively. Similarly, in Thalassiosira pseudonana, TpSLC4-2 (SFS-FAP) and
TpSLC4-3 (VNA-FPT) have been identified as candidate chloroplast membrane
HCO 3 - transporters. The pre-sequences in diatoms significantly differ from thosepresent in green algae or land plants as they possess a bipartite sequencecomprising of a signal peptide sequence along with transit peptide.

2Despite the advances in the field of diatom research in recent years, the function of
the multi-layered chloroplast envelope membranes in diatoms is still largely
unexplored at the molecular level and the candidate proteins involved in the entry of
HCO 3 - into the stroma requires experimental validation.
Another area of recent interest is the recently discovered pyshell proteins. The
confocal analysis of these proteins showed that these proteins were present on thesurface of the pyrenoid in chloroplast (proposed by previous studies conducted in lab)of P.tricornutum and T. pseudonana. Although exact function of these proteins is notknown any addition in this knowledge could be a stepping stone in proposition ofcarbon uptake model in these unicellular photosynthetic organisms.
Since studying the function of proteins through mutagenesis has been widely used
these days, an attempt was made to knockout the candidate transporter proteins (inboth P.tricornutum and T. pseudonana) and the pyshell proteins (in T. pseudonana)and then study their effects on the mutants. The genes chosen for the study have been
described in the table below (Table no.1).
SerialGene nameProtein id in JGIGenomic location

1.

Ptslc4-7

45656(Phatr2)

chr_7:834212-836081 (-)

2.

Ptslc4-6

43194(Phatr2)

Chr_1:2279932-2281914 (-)

3.

Tpslc4-2

260936 (Thaps3)

chr_1:1419165-1419927 (+)

4.

Tpslc4-3

267979 (Thaps3)

chr_1:974609-977268 (-)

5.

Tp7881(Tpphyshell1)7881(Thaps3)

chr_9:155342-156226 (-)

6.

Tp23918(Tpphyshell2)23918(Thaps3)

chr_9:157777-159128 (+)

7.

Tp7883(Tpphyshell3)7883(Thaps3)

chr_9:159916-160818 (+)

no.

Table no.1: Genes used for the functional characterization.
For this, CAS9 nickase approach was used (Figure no.2) and guide RNA pairs wereshortlisted using the benchling software (https://www.benchling.com/crispr/).

Figure 2: CRISPR/Cas9 nickase system

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The guide RNA pair with the out of frame score more than 66 (calculated using thecas-offinder tool (http://www.rgenome.net/cas-offinder/) and those having the least
off-target activity were finally used for synthesis of constructs. The primer pair used
for the amplification of gRNA for each gene have been depicted in the table below(Table no.2).
S. No.

Primer name

Sequence used (5’to 3’) for the initial
amplification of gRNA1.

Fptslc7

AGGTCTCATCGAGGTTGATCCGATGATTGTCAAGTTTAAGAGCTAGAAAT

2.

Rptslc7

GGGTCTCTAAACATTGTGCGAGACAGGCTACGCTCGACTTTGAAGGTGTT

3.

Fptslc6

AGGTCTCATCGAGACTGACCGCCGGAGAAAGACGTTTAAGAGCTAGAAAT

4.

Rptslc6

GGGTCTCTAAACTGCGTTATCTCCGAAGCAATCTCGACTTTGAAGGTGTT

5.

Ftpslc3

AGGTCTCATTGTGAAACCCACCCACGCATATACGTTTAAGAGCTAGAAAT

6.

Rtpslc3

GGGTCTCTAAACAGAATCATCATAAAGGCACTCACAATTTCGGCAAAACG

7.

Ftpslc2

AGGTCTCATTGTGCATGGTACCGATCGCCCCATGTTTAAGAGCTAGAAAT

8.

Rtpslc2

GGGTCTCTAAACCACATTGCTGTAGAACTGACCACAATTTCGGCAAAACG

9.

Ftppyshell1

AGGTCTCATTGTGAGTTGTCCCAGATCTCGGCAGTTTAAGAGCTAGAAAT

10.

Rtppyshell1

GGGTCTCTAAACCCCTGCACAATCACGGGGGACACAATTTCGGCAAAACG

11.

Ftppyshell2

AGGTCTCATTGTGTAACGGCATTGAAGGTACGAGTTTAAGAGCTAGAAAT

12.

Rtppyshell2

GGGTCTCTAAACCGGTGTTGGGGCCGCGGGGACACAATTTCGGCAAAACG

13.

Ftppyshell3

AGGTCTCATTGTGATCAATGGATGGGTTGGCGAGTTTAAGAGCTAGAAAT

14.

Rtppyshell3

GGGTCTCTAAACTCTTGAGCAACACTTGCACTCACAATTTCGGCAAAACG

Table no. 2: The primer sequences used for gRNA amplification from the templatevector.
The gRNAs were cloned in the insertion vector using BsaI restriction enzyme sites.
The methodology followed for cloning and the vector used have been illustrated in the
figure below (Figure3A and 3B).

A

B

Figure 3(A): Strategy for construction of recombinant vector with dual sgRNAs.
Step1: Amplification of the dual sgRNA cassette from the PCR template vector by
PCR with specific gRNAs with BsaI restriction site. Step2: Digestion of the insertion
vector and PCR amplified dual sgRNA cassette by BsaI. Step3: Ligation of theinsertion vector and dual sgRNA cassette.
4Figure 3(B): A schematic depiction of two vector expression system of dual sgRNAsand Cas9 nickase (D10A). A cas9 nickase D10A contains a point mutation thatconverts a GAC codon to GCC in the amino acid sequence coding for the diacas9
gene. The fragment encoding Cas9 nickase (D10A) was cloned into the pNR vector toproduce pNR_Cas9nD10A vector. The dual sgRNA vector is the expression vector ofthe two sgRNA driven by the U6 promoter.
The clones were confirmed by sequencing. The vector harboring the gRNA pair alongwith the nickase vector were co-transformed into the T. pseudonana cells (O.D. 7 3 0 0.1 to 0.2) by particle bombardment method. The cells were then screened on plates
containing F/2 medium (containing NH 4 + as nitrogen source) with nourseothricin. Thecolonies that appeared on the plates were further streaked on F/2 medium (containingNO 3 - as nitrogen source).
The total number of colonies obtained were 38 (for T.pseudonana). No colonies wereobserved after transformation of P. tricornutum, which reflects failure of thetransformation process or the lethality of the knock out mutant. The maximum numberof colonies were observed in Tpphyshell2 (16 colonies) followed by Tppyshell3 (12colonies) and Tpslc4-2 and Tppyshell1 (5 colonies each). No colonies were observedin Tpslc4-3 transformed plates.
An active transport of carbon from the cytoplasm into the chloroplast is the main
driver of the diatom CCM. Therefore, the identification of the chloroplast locatedbicarbonate transporters is crucial step for the understanding of a diatomic CCM. ThePtSLC4-6 and PtSLC4-7 genes have been reported to be constitutively expressed
under high and low CO 2 conditions suggesting that these transporters regulate DIC
flow from the cytosol to the plastid regardless of ambient CO 2 concentrations(Nakajima et al., 2013). If this is the case, then a knock out mutant of thesetransporters may be lethal. And if the activity of either of them is perturbed, theorganism may still survive as the other may carry out the transport function. It is also
expected that the loss of the chloroplast pump may result in higher cytoplasmic DIC
which may increase the CO 2 leakage.
The effect of mutagenesis on the pyshell proteins is an unknown facet of CCM
research. The role of these protein in carbon sequestration and the reason for theexistence of the protein in close vicinity of other CCM components in the pyrenoids isstill a fascinating question.

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Deadline： W ithin two months af ter f inishing the research pe riod.
Sabbatical leave with grant: Sub mit this report t o President with conf irmation by the dean ofschool you belong to.
※ Postdoctoral f ellow is required to sub mit this report with conf irmation by the de an of graduate

school bef ore the end of emplo yment pe riod.
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